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Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes the RNA/DNA primer associated with Okazaki fragments in DNA replication. Here, crystals of the complex between the catalytic domain of human FEN1 and a DNA product have been obtained. For efficient crystallization screening, a DNA-protein complex crystallization screening (DPCS) kit was designed based on commercial crystallization kits. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 61.0, b = 101.3, c = 106.4 A, beta = 106.4 degrees. The asymmetric unit is predicted to contain two complexes in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 2.75 A.

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3'-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.

Background: Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase delta. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied., Results: Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA., Conclusion: These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms twoß-strands connected by a short helix, theßA-aA-ßB motif, participating inß-ßand hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive‘locked-down’orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation. [ABSTRACT FROM AUTHOR]

A single-chain antibody specific for the pyrimidine(6-4)pyrimidone photoproduct in DNA was constructed and its binding properties to the cognate oligonucleotides were investigated. The fluorescent property of 2-aminopurine was used to study the binding mode of the antibody fragment to the pyrimidine(6-4)pyrimidone photoproduct in double-stranded DNA. The results indicated that the single-chain antibody recognizes the lesion in the single-stranded state. [ABSTRACT FROM AUTHOR]