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Temporal and spatial regulation of the replication factory is important for efficient DNA replication. However, the underlying molecular mechanisms are not well understood. Here, we report that ATAD5 regulates the lifespan of replication factories. Reduced expression of ATAD5 extended the lifespan of replication factories by retaining proliferating cell nuclear antigen (PCNA) and other replisome proteins on the chromatin during and even after DNA synthesis. This led to an increase of inactive replication factories with an accumulation of replisome proteins. Consequently, the overall replication rate was decreased, which resulted in the delay of S-phase progression. Prevalent detection of PCNA foci in G2 phase cells after ATAD5 depletion suggests that defects in the disassembly of replication factories persist after S phase is complete. ATAD5-mediated regulation of the replication factory and PCNA required an intact ATAD5 ATPase domain. Taken together, our data imply that ATAD5 regulates the cycle of DNA replication factories, probably through its PCNA-unloading activity.

A novel, genetic immunodeficiency syndrome has been recently described, herein termed "MonoMAC". It is characterized by severe circulating monocytopenia, NK- and B-lymphocytopenia, severe infections with M. avium complex (MAC), and risk of progression to myelodysplasia/acute myelogenous leukemia. Detailed bone marrow analyses performed on 18 patients further define this disorder. The majority of patients had hypocellular marrows with reticulin fibrosis and multilineage dysplasia affecting the myeloid (72%), erythroid (83%) and megakaryocytic (100%) lineages. Cytogenetic abnormalities were present in 10 of 17 (59%). Despite B-lymphocytopenia, plasma cells were present but were abnormal (e.g. CD56(+)) in nearly half of cases. Increased T-cell large granular lymphocyte populations were present in 28% of patients. Chromosomal breakage studies, cell cycle checkpoint functions, and sequencing of TERT and K-RAS genes revealed no abnormalities. MonoMAC appears to be a unique, inherited syndrome of bone marrow failure. We describe distinctive bone marrow features to help in its recognition and diagnosis. (Clinicaltrials.gov identifiers: NCT00018044, NCT00923364, NCT01212055).

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis., Competing Interests: The authors have declared that no competing interests exist.

Chromosome integrity in response to chemically or radiation-induced chromosome breaks and the perturbation of ongoing replication forks relies on multiple DNA repair mechanisms. However, repair of these lesions may lead to unwanted chromosome rearrangement if not properly executed or regulated. As these types of chromosomal alterations threaten the cell's and the organism's very own survival, multiple systems are developed to avoid or at least limit break-induced chromosomal rearrangements. In this review, we highlight cellular strategies for repressing DNA break-induced chromosomal translocations in multiple model systems including yeast, mouse, and human. These pathways select proper homologous templates or broken DNA ends for the faithful repair of DNA breaks to avoid undesirable chromosomal translocations.

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.

The temperature-sensitive phenotypes of yku70Delta and yku80Delta have provided a useful tool for understanding telomere homeostasis. Mutating the helicase domain of the telomerase inhibitor Pif1 resulted in the inactivation of cell cycle checkpoints and the subsequent rescue of temperature sensitivity of the yku70Delta strain. The inactivation of Pif1 in yku70Delta increased overall telomere length. However, the long G-rich, single-stranded overhangs at the telomeres, which are the major cause of temperature sensitivity, were slightly increased. Interestingly, the rescue of temperature sensitivity in strains having both pif1-m2 and yku70Delta mutations depended on the homologous recombination pathway. Furthermore, the BLM/WRN helicase yeast homolog Sgs1 exacerbated the temperature sensitivity of the yku70Delta strain. Therefore, the yKu70-80 heterodimer and telomerase maintain telomere size, and the helicase activity of Pif1 likely also helps to balance the overall size of telomeres and G-rich, single-stranded overhangs in wild-type cells by regulating telomere protein homeostasis. However, the absence of yKu70 may provide other proteins such as those involved in homologous recombination, Sgs1, or Pif1 additional access to G-rich, single-stranded DNA and may determine telomere size, cell cycle checkpoint activation, and, ultimately, temperature sensitivity.

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.

The inactivation of either subunit of the Ku70-Ku80 heterodimer, which functions in nonhomologous end-joining and telomere maintenance, generates severe defects such as sensitivity to DNA damage, telomere shortening, and increased gross chromosomal rearrangements (GCRs) that are frequently observed in many cancers. To understand the mechanism of Ku as a genome gatekeeper, we overexpressed the yKu70-yKu80 heterodimer and monitored the formation of GCRs. Ku overexpression suppressed the formation of either spontaneously generated GCRs or those induced by treatments with different DNA damaging agents. Interestingly, this suppression depended on Ku's interaction with DNA damage checkpoints and not through nonhomologous end-joining. We also demonstrate that the inactivation of telomerase inhibitor, Pif1 along with Ku overexpression or the overexpression of Pif1 in either yku70 or yku80 strains arrested the cell cycle at S phase in a DNA damage checkpoint-dependent fashion. Lastly, Ku overexpression causes cell growth delay, which depends on intact Rad27. In summary, the results presented here suggest that Ku functions as a genomic gatekeeper through its crosstalk with DNA damage checkpoints.

The accumulation of gross chromosomal rearrangements (GCRs) is characteristic of cancer cells. Multiple pathways that prevent GCRs, including S-phase cell cycle checkpoints, homologous recombination, telomere maintenance, suppression of de novo telomere addition, chromatin assembly, and mismatch repair, have been identified in Saccharomyces cerevisiae. However, pathways that promote the formation of GCRs are not as well understood. Of these, the de novo telomere addition pathway and nonhomologous end-joining are the best characterized. Here, we demonstrate that defects in the mitotic checkpoint and the mitotic exit network can suppress GCRs in strains containing defects that increase the GCR rate. These data suggest that functional mitotic checkpoints can play a role in the formation of genome rearrangements.

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.

Cancer cells show increased genome rearrangements, although it is unclear what defects cause these rearrangements. Previous studies have implicated the Saccharomyces cerevisiae replication checkpoint in the suppression of spontaneous genome rearrangements. In the present study, low doses of methyl methane sulfonate that activate the intra-S checkpoint but not the G1 or G2 DNA damage checkpoints were found to cause increased accumulation of genome rearrangements in both wild-type strains and to an even greater extent in strains containing mutations causing defects in the intra-S checkpoint. The rearrangements were primarily translocations or events resulting in deletion of a portion of a chromosome arm along with the addition of a new telomere. Combinations of mutations causing individual defects in the RAD24 or SGS1 branches of the intra-S checkpoint or the replication checkpoint showed synergistic interactions with regard to the spontaneous genome instability rate. PDS1 and the RAD50-MRE11-XRS2 complex were found to be important members of all the S-phase checkpoints in suppressing genome instability, whereas RAD53 only seemed to play a role in the intra-S checkpoints. Combinations of mutations that seem to result in inactivation of the S-phase checkpoints and critical effectors resulted in as much as 12,000-14,000-fold increases in the genome instability rate. These data support the view that spontaneous genome rearrangements result from DNA replication errors and indicate that there is a high degree of redundancy among the checkpoints that act in S phase to suppress such genome instability.