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DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Altered telomere maintenance mechanism (TMM) is linked to increased DNA damage at telomeres and telomere uncapping. We previously showed that HIV-1 latent cells have altered TMM and are susceptible to ligands that target G-quadruplexes (G4) at telomeres. Susceptibility of latent cells to telomere targeting could potentially be used to support approaches to eradicate HIV reservoirs. However, G4 ligands also target G-quadruplexes in promoters blocking gene transcription. Since HIV promoter sequence can form G-quadruplexes, we investigated whether G4 ligands interfere with HIV-1 promoter activity and virus reactivation from latency, and whether telomere targeting could be combined with latency reversing agents (LRAs) to promote elimination of HIV reservoirs. Our results indicate that Sp1 binding region in HIV-1 promoter can adopt G4 structures in duplex DNA, and that in vitro binding of Sp1 to G-quadruplex is blocked by G4 ligand, suggesting that agents targeting telomeres interfere with virus reactivation. However, our studies show that G4 agents do not affect HIV-1 promoter activity in cell culture, and do not interfere with latency reversal. Importantly, primary memory CD4 + T cells infected with latent HIV-1 are more susceptible to combined treatment with LRAs and G4 ligands, indicating that drugs targeting TMM may enhance killing of HIV reservoirs. Using a cell-based DNA repair assay, we also found that HIV-1 infected cells have reduced efficiency of DNA mismatch repair (MMR), and base excision repair (BER), suggesting that altered TMM in latently infected cells could be associated with accumulation of DNA damage at telomeres and changes in telomeric caps.

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC 50 ) for BCV and a 1.9-fold increased EC 50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity., (Copyright © 2018 American Society for Microbiology.)

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.

Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3.

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

SAMHD1 (SAM domain- and HD domain-containing protein 1) is a dGTP-dependent dNTP triphosphohydrolase that converts dNTPs into deoxyribonucleosides and triphosphates. Therefore, SAMHD1 expression, particularly in non-dividing cells, can restrict retroviral infections such as HIV and simian immunodeficiency virus by limiting cellular dNTPs, which are essential for reverse transcription. It has previously been established that dGTP acts as both an activator and a substrate of this enzyme, suggesting that phosphohydrolase activity of SAMHD1 is regulated by dGTP availability in the cell. However, we now demonstrate biochemically that the NTP GTP is equally capable of activating SAMHD1, but GTP is not hydrolyzed by the enzyme. Activation of SAMHD1 phosphohydrolase activity was tested under physiological concentrations of dGTP or GTP found in either dividing or non-dividing cells. Because GTP is 1000-fold more abundant than dGTP in cells, GTP was able to activate the enzyme to a greater extent than dGTP, suggesting that GTP is the primary activator of SAMHD1. Finally, we show that SAMHD1 has the ability to hydrolyze base-modified nucleotides, indicating that the active site of SAMHD1 is not restrictive to such modifications, and is capable of regulating the levels of non-canonical dNTPs such as dUTP. This study provides further insights into the regulation of SAMHD1 with regard to allosteric activation and active site specificity.

The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for treating HIV/AIDS. We found that polymerization activity of a reverse transcriptase (RT) with the E478Q mutation that inactivates the RNase H catalytic site is much more sensitive to efavirenz than wild-type RT, indicating that a functional RNase H attenuates the effectiveness of efavirenz. Moreover, efavirenz actually stimulated wild-type RNase H binding and catalytic functions, indicating another link between efavirenz action and RNase H function. During reverse transcription in vivo, the RT that is extending the DNA primer also periodically cleaves the genomic RNA. The RNase H makes primary template cuts ~18 nucleotides from the growing DNA 3'-end, and when the RT pauses synthesis, it shifts to make secondary cuts ~9 nucleotides from the DNA 3'-end. After synthesis, RTs return to bind the remaining template RNA segments at their 5'-ends and make primary and secondary cuts, 18 and 9 nucleotides in, respectively. We found that efavirenz stimulates both 3'- and 5'-directed RNase H activity. Use of specific substrates revealed a particular acceleration of secondary cuts. Efavirenz specifically promoted binding of the RT to RNase H substrates, suggesting that it stabilizes the shifting of RTs to make the secondary cuts. We further showed that efavirenz similarly stimulates the RNase H of an RT from a patient-derived virus that is highly resistant and grows more rapidly in the presence of low concentrations of efavirenz. We suggest that for efavirenz-resistant RTs, stimulated RNase H activity contributes to increased viral fitness.

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.

The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are ~1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

First discovered as a structure-specific endonuclease that evolved to cut at the base of single-stranded flaps, flap endonuclease (FEN1) is now recognized as a central component of cellular DNA metabolism. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. For Okazaki fragments, the RNA primer is displaced into a 5' flap and then cleaved off. FEN1 binds to the flap base and then threads the 5' end of the flap through its helical arch and active site to create a configuration for cleavage. The threading requirement prevents this active nuclease from cutting the single-stranded template between Okazaki fragments. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. Overall, recent advances in our knowledge of FEN1 suggest that it was an ancient protein that has been fine-tuned over eons to coordinate many essential DNA transactions.

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase β in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.

Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (k(on)) for template binding and a lower equilibrium binding constant K(d) for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k(off) values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.

Steps in the replication of human immunodeficiency virus type 1 (HIV-1) occurring in the virus but not in the host are preferred targets of antiretroviral therapy. Strand transfer is unique; the DNA strand being made by viral reverse transcriptase (RT) is moved from one RNA template position to another. Understanding the mechanism requires knowing whether the RT directly mediates the template exchange or dissociates during the exchange, so that it occurs by polymer dynamics. Earlier work in vitro showed that the presence of an RT-trapping polymer would allow synthesis on the original or donor template but completely block transfer and subsequent synthesis on the second or acceptor template. One interpretation is that the RT must dissociate during transfer, but an alternative is that sequestration of non-polymerizing RTs prevents polymerization-independent ribonuclease H (RNase H) cleavages of the donor template necessary for strand exchange. To resolve this ambiguity, we designed a primer-template system that allows strand transfer without RNase H activity. Using an RNase H negative mutant RT, we showed that a polymer trap still prevented strand transfer. This confirms that RT dissociates during strand transfer. The presence of HIV-1 nucleocapsid protein, which promotes strand exchange, had little effect on this outcome. Additional assays showed that both the wild-type RT and a multiple nucleoside RT inhibitor-resistant HIV-1 RT containing an extended fingers domain, which is characterized by its enhanced primer-template binding affinity, were unable to transfer with the trapping polymer. This implies that common sequence variations among RTs are unlikely to alter dissociation feature., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A preferred site for HIV-1 recombination was identified in vivo and in vitro surrounding the beginning of the HIV-1 gag gene. This G-rich gag hotspot for recombination contains three evenly spaced G-runs that stalled reverse transcriptase. Disruption of the G-runs suppressed both the associated pausing and strand transfer in vitro. Significantly, this same gag sequence was able to fold into a G-quartet monomer, dimer, and tetramer, depending on the cations employed. The pause band at the G-run (nucleotide (nt) 405-409), which was predicted to be involved in forming a G-quartet monomer, diminished with increased HIV-1 nucleocapsid (NC) protein. More NC induced stronger pauses at other G-runs (nt 363-367 and nt 382-384), a region that forms a G-quartet dimer, adhering the two RNA templates. We hypothesized that NC induces the unfolding of the monomeric G-quartet but stabilizes the dimeric interaction. We tested this by inserting a known G-quartet formation sequence, 5'-(UGGGGU)(4)-3', into a relatively structure-free template from the HIV-1 pol gene. Strand transfer assays were performed with cations that either encourage (K(+)) or discourage (Li(+)) G-quartet formation with or without NC. Strikingly, a G-quartet monomer was observed without NC, whereas a G-quartet dimer was observed with NC, both only in the presence of K(+). Moreover, the transfer efficiency of the dimerized template (with K(+) and NC) reached about 90%, approximately 2.5-fold of that of the non-dimerized template. Evidently, template dimerization induced by NC creates a proximity effect, leading to the unique high peak of transfer at the gag recombination hotspot.

DNA damage, stalled replication forks, errors in mRNA splicing, and availability of nutrients activate specific phosphatidylinositiol-3 kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2, or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double-strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA.

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5' single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5' single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.

After HIV-1 enters a human cell, its RNA genome is converted into double stranded DNA during the multistep process of reverse transcription. First (minus) strand DNA synthesis is initiated near the 5' end of the viral RNA, where only a short fragment of the genome is copied. In order to continue DNA synthesis the virus employs a complicated mechanism, which enables transferring of the growing minus strand DNA to a remote position at the genomic 3' end. This is called minus strand DNA transfer. The transfer enables regeneration of long terminal repeat sequences, which are crucial for viral genomic DNA integration into the host chromosome. Numerous factors have been identified that stimulate minus strand DNA transfer. In this review we focus on describing protein-RNA and RNA-RNA interactions, as well as RNA structural features, known to facilitate this step in reverse transcription.

A tRNA gene-like sequence has been identified near the 3' end of HIV-1. Two segments of this sequence (motif 9 and segment 1) promoted minus strand transfer in vitro. The segments are complementary to the tRNA(3)(Lys) primer, and apparently act by binding the tRNA, thereby bringing the 3' and 5' ends of viral RNA into proximity for strand transfer. In this report, we used full-length HIV-1 to demonstrate biological relevance of these segments. We constructed HIV-1 genomes capable of single cycle infection and altered in one or both of two segments. We devised a real time PCR method for quantifying the amount of (-)ssDNA that completes transfer. Results showed that depending on the mutation the efficiency of transfer decreased from 9% to 26%. Alteration of segment 1 had the greatest effect. Alteration of motif 9 or both sequences also caused a reduction, but smaller than alteration of segment 1 alone., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo.

Dna2 endonuclease/helicase participates in eukaryotic DNA transactions including cleavage of long flaps generated during Okazaki fragment processing. Its unusual substrate interaction consists of recognition and binding of the flap base, then threading over the 5'-end of the flap, and cleaving periodically to produce a terminal product ∼5 nt in length. Blocking the 5'-end prevents cleavage. The Dna2 ATP-driven 5' to 3' DNA helicase function promotes motion of Dna2 on the flap, presumably aiding its nuclease function. Here we demonstrate using two different nuclease-dead Dna2 mutants that on substrates simulating Okazaki fragments, Dna2 must thread onto an unblocked 5' flap to display helicase activity. This requirement is maintained on substrates with single-stranded regions thousands of nucleotides in length. To our knowledge this is the first description of a eukaryotic helicase that cannot load onto its tracking strand internally but instead must enter from the end. Biologically, the loading requirement likely helps the helicase to coordinate with the Dna2 nuclease function to prevent creation of undesirably long flaps during DNA transactions.

FEN1 cleaves 5' flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5'-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5'-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5'-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.

Reconstitution of eukaryotic Okazaki fragment processing implicates both one- and two-nuclease pathways for processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway.

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of multidrug therapy for HIV-1. Understanding the effect of NNRTI-resistant mutants on virus replication and reverse transcriptase (RT) function is valuable for the development of extended-spectrum NNRTIs. We measured the fitness of six NNRTI-resistant mutants, the K103N, V106A, Y181C, G190A, G190S, and P236L viruses, using a flow cytometry-based cell culture assay. K103N and Y181C viruses had fitness similar to that of the wild type while V106A, G190A, G190S, and P236L viruses had reduced fitness. We also determined the biochemical correlates of fitness by measuring the RNase H and polymerization activities of recombinant mutant RTs and virion-associated RTs. The RNase H activities of recombinant and virion-associated RTs correlated with the relative fitness for each mutant. K103N and Y181C mutants had normal RNase H activity; V106A, G190A, and G190S mutants had moderate reductions in activity; and the P236L mutant had substantially reduced activity. With the exception of the P236L mutant, reduced fitness correlates with low virion-associated polymerization efficiency and reduced RT content. Reduced polymerase function in virions derived from low RT content rather than an intrinsic polymerization defect in each RT protein. In conclusion, severe defects in RNase H activity alone, exemplified by the P236L mutant, appear sufficient to cause a substantial reduction in fitness. For the other NNRTI mutants, reductions in RT content decreased both polymerization and RNase H activity in virions. RNase H reduction was compounded by intrinsic RNase H defects in the mutant RTs.

Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase delta (pol delta), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/nuclease contribute to the maturation process by elongating the flap displaced by pol delta. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review, we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5'-3' endonuclease, the 5'-3' helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase alpha.

We identified a sequence embedded in the U3-R region of HIV-1 RNA that is highly complementary to human tRNA(3)(Lys). The free energy of annealing to tRNA(3)(Lys) is significantly lower for this sequence and the primer-binding site than for other viral sequences of similar length. The only interruption in complementarity is a 29-nucleotide segment inserted where a tRNA intron would be expected. The insert contains the TATA box for viral RNA transcription. The embedded sequence includes a 9-nucleotide segment previously reported to aid minus-strand transfer by binding the primer tRNA(3)(Lys). Reconstituting transfer in vitro, we show that including segments from the embedded sequence in the acceptor template, beyond the 9 nucleotides, further increases transfer efficiency. We propose that a gene encoding tRNA(3)(Lys) was incorporated during HIV-1 evolution and retained, largely intact, because of its roles in transcription and strand transfer.

Dna2 is a nuclease/helicase with proposed roles in DNA replication, double-strand break repair and telomere maintenance. For each role Dna2 is proposed to process DNA substrates with a 5'-flap. To date, however, Dna2 has not revealed a preference for binding or cleavage of flaps over single-stranded DNA. Using DNA binding competition assays we found that Dna2 has substrate structure specificity. The nuclease displayed a strong preference for binding substrates with a 5'-flap or some variations of flap structure. Further analysis revealed that Dna2 recognized and bound both the single-stranded flap and portions of the duplex region immediately downstream of the flap. A model is proposed in which Dna2 first binds to a flap base, and then the flap threads through the protein with periodic cleavage, to a terminal flap length of approximately 5 nt. This resembles the mechanism of flap endonuclease 1, consistent with cooperation of these two proteins in flap processing.

Human immunodeficiency virus-1 employs strand transfer for recombination between two viral genomes. We have previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long-distance propagation of the hybrid. Evaluation was performed on a unique substrate, with a short gap serving as a precreated invasion site. In our current work, this substrate provided an opportunity for us to test what factors influence a specific invasion site to support transfer, and to distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or polymerization-independent mode. Polymerization-dependent and polymerization-independent RNase H were found to be important in creating efficiently used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a precreated invasion site in the presence of NC, were stimulated by polymerization-dependent, but not polymerization-independent, RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer even if synthetic progress of reverse transcriptase was intentionally slowed, consistent with strand exchange by random walk in which rate declines precipitously with distance.

The co-packaged RNA genomes of human immunodeficiency virus-1 recombine at a high rate. Recombination can mix mutations to generate viruses that escape immune response. A cell-culture-based system was designed previously to map recombination events in a 459-bp region spanning the primer binding site through a portion of the gag protein coding region. Strikingly, a strong preferential site for recombination in vivo was identified within a 112-nucleotide-long region near the beginning of gag. Strand transfer assays in vitro revealed that three pause bands in the gag hot spot each corresponded to a run of guanosine (G) residues. Pausing of reverse transcriptase is known to promote recombination by strand transfer both in vivo and in vitro. To assess the significance of the G runs, we altered them by base substitutions. Disruption of the G runs eliminated both the associated pausing and strand transfer. Some G-rich sequences can develop G-quartet structures, which were first proposed to form in telomeric DNA. G-quartet structure formation is highly dependent on the presence of specific cations. Incubation in cations discouraging G-quartets altered gel mobility of the gag template consistent with breakdown of G-quartet structure. The same cations faded G-run pauses but did not affect pauses caused by hairpins, indicating that quartet structure causes pausing. Moreover, gel analysis with cations favoring G-quartet structure indicated no structure in mutated templates. Overall, results point to reverse transcriptase pausing at G runs that can form quartets as a unique feature of the gag recombination hot spot.

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase delta was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase delta to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase beta (pol beta), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol beta is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol beta or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol beta with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.

Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: (1) invasion of the initial or donor primer template by the second or acceptor template; (2) propagation of the primer-acceptor hybrid; and (3) primer terminus transfer. Invasion occurs at a site at which the reverse transcriptase ribonuclease H (RNase H) has created a nick or short gap in the donor template. We used biochemical reconstitution to determine the distance over which a single invasion site can promote transfer. The DNA-primed RNA donor template used had a single-stranded pre-created invasion site (PCIS). Results showed that the PCIS could influence transfer by 20 or more nucleotides in the direction of synthesis. This influence was augmented by viral nucleocapsid protein and additional reverse transcriptase-RNase H cleavage. Strand-exchange assays were performed specifically to assess the distance over which a hybrid interaction initiated at the PCIS could propagate to achieve transfer. Propagation by simple branch migration of strands was limited to 24-32 nt. Additional RNase H cuts in the donor RNA allowed propagation to a maximum distance of 32-64 nt. Overall, results indicate that a specific invasion site has a limited range of influence on strand transfer. Evidently, a series of invasion sites cannot collaborate over a long distance to promote transfer. This result explains why the frequency of recombination events does not increase with increasing distance from the start of synthesis, a characteristic that supports effective mixing of viral mutations.

Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5'-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing.

Donor-acceptor template systems in vitro were designed to test mechanisms of minus strand transfer of human immunodeficiency virus 1 (HIV-1). Donor RNA D199, extending from the 5' end of the HIV-1 genome to the primer binding site (PBS), promoted transfer to only 35% with an acceptor RNA representing the 3' terminal 97 nucleotides, whereas donor RNA D520, including an additional 321 nucleotides 3' of PBS, exhibited 75% transfer. Both donors transferred through an invasion-driven pathway, but transfer was stimulated by the folding structure resulting from the extra segment in D520. In this study, the significance of interaction between the tRNA(lys3) primer and U3 was examined. Measurements utilizing acceptors having or lacking the U3 region complementary with tRNA(lys3) indicated that a tRNA(lys3)-U3 interaction compensated for inefficient acceptor invasion observed with D199. Stimulation presumably occurred because binding to tRNA(lys3) increased the proximity of the acceptor to elongated cDNA, improving transfer to 78% efficiency with D199, and even higher to 85% with D520. The stimulation did not require natural viral sequences but could be achieved by substituting the original U3 sequence with an equal length sequence that binds a different region of tRNA(lys3). Comparison between acceptors sharing the natural region for tRNA(lys3)-U3 interaction but having or lacking the acceptor invasion site demonstrated that tRNA(lys3)-U3 interaction and acceptor invasion cooperate for maximal stimulation. Overall, observations suggest that both proximity and invasion mechanisms are applied successively by HIV-1 for efficient minus strand transfer.

Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase delta displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA-binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Because Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.

Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability.

Genotoxic stress activates the phosphatidylinositol 3-kinase-like kinases (PIKKs) that phosphorylate proteins involved in cell cycle arrest, DNA repair and apoptosis. Previous work showed that the PIKK ataxia telangiectasia mutated (ATM) but not ATM and Rad3 related phosphorylates p53 (Ser15) during hyperoxia, a model of prolonged oxidative stress and DNA damage. Here, we show hSMG-1 is responsible for the rapid and early phosphorylation of p53 (Ser15) and that ATM helps maintain phosphorylation after 24 h. Despite reduced p53 phosphorylation and abundance in cells depleted of hSMG-1 or ATM, levels of the p53 target p21 were still elevated and the G(1) checkpoint remained intact. Conditional overexpression of p21 in p53-deficient cells revealed that hyperoxia also stimulates wortmannin-sensitive degradation of p21. siRNA depletion of hSMG-1 or ATM restored p21 stability and the G(1) checkpoint during hyperoxia. These findings establish hSMG-1 as a proximal regulator of DNA damage signaling and reveal that the G(1) checkpoint is tightly regulated during prolonged oxidative stress by both PIKK-dependent synthesis and proteolysis of p21.

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses replicate through reverse transcription, a process in which the single stranded RNA of the viral genome is converted to a double stranded DNA. The virally encoded reverse transcriptase (RT) mediates reverse transcription through DNA polymerase and RNase H activities. Conversion of the plus strand RNA to plus/minus strand RNA/DNA hybrid involves a transfer of the growing DNA strand from one site on the genomic RNA to another. This is called minus strong-stop DNA transfer. Later synthesis of the second or plus DNA strand involves a second strand transfer, involving a similar mechanism as the minus strand transfer. A basic feature of the strand transfer mechanism is the use of the RT RNase H to remove segments of the RNA template strand from the growing DNA strand, freeing a single stranded region to anneal to the second site. Viral nucleocapsid protein (NC) functions to promote transfer by facilitating this strand exchange process. Two copies of the RNA genomes, sometimes non-identical, are co-packaged in the genomes of retroviruses. The properties of the reverse transcriptase allow a transfer of the growing DNA strand between these genomes to occur occasionally at any point during reverse transcription, producing recombinant viral progeny. Recombination promotes structural diversity of the virus that helps it to survive host immunity and drug therapy. Recombination strand transfer can be forced by a break in the template, or can occur at sites where folding structure of the template pauses the RT, allowing a concentration of RNase H cleavages that promote transfers. Transfer can be a simple one-step process, or can proceed by a complex multi-step invasion mechanism. In this latter process, the second RNA template interacts with the growing DNA strand well behind the DNA 3'-terminus. The newly formed RNA-DNA hybrid expands by branch migration and eventually catches the elongating DNA primer 3'-terminus to complete the transfer. Transfers are also promoted by interactions between the two RNA templates, which accelerate transfer by a proximity effect. Other details of the role of strand transfers in reverse transcription and the biochemical features of the transfer reaction are discussed.

Estrogen hormone 17beta-estradiol (E(2)) is involved in the physiology and pathology of many tissues. E(2) information is conveyed by the transcription factors estrogen receptors (ER) alpha and beta that mediate a complex array of nuclear and non-nuclear events. The interaction of ER with specific DNA sequences, estrogen-responsive elements (EREs), constitutes a critical nuclear signaling pathway. In addition, E(2)-ER regulates transcription through interactions with transfactors bound to their cognate regulatory elements on DNA, hence the ERE-independent signaling pathway. However, the relative importance of the ERE-independent pathway in E(2)-ERbeta signaling is unclear. To address this issue, we engineered an ERE-binding defective ERbeta mutant (ERbeta(EBD)) by changing critical residues in the DNA-binding domain required for ERE binding. Biochemical and functional studies revealed that ERbeta(EBD) signaled exclusively through the ERE-independent pathway. Using the adenovirus infected ER-negative cancer cell models, we found that although E(2)-ERbeta(EBD) regulated the expression of a number of genes identified by microarrays, it was ineffective in altering cellular proliferation, motility, and death in contrast to E(2)-ERbeta. Our results indicate that genomic responses from the ERE-independent pathway to E(2)-ERbeta are not sufficient to alter the cellular phenotype. These findings suggest that the ERE-dependent pathway is a required signaling route for E(2)-ERbeta to induce cellular responses.