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 two b-strands connected by a short helix, the bA-aA-bB motif, participating in b-b and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21 CIP1/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.

It has been shown previously that deoxyguanosine residues in DNA are hydroxylated at the C-8 position both in vitro and in vivo to produce 8-hydroxydeoxyguanosine (8-OH-dG) by various agents that produce oxygen radicals such as reducing reagents-O2, metal ions-O2, polyphenol-H2O2-Fe3+, asbestos-H2O2 or ionizing radiation. These agents are mostly either mutagenic or carcinogenic; therefore, the formation of 8-OH-dG can also be considered a likely cause of mutation or carcinogenesis by oxygen radicals. It is of interest to know whether the 8-OH-dG residue in DNA is misread during DNA replication. To answer this question, we have examined the effect of the 8-OH-dG residue in DNA on the fidelity of DNA replication using a DNA synthesis system in vitro with Escherichia coli DNA polymerase I (Klenow fragment). The synthetic oligodeoxynucleotides, with or without an 8-OH-dG residue in a specified position, were chemically synthesized and used as templates for DNA synthesis under the conditions of the dideoxy chain termination sequencing method. Surprisingly, in addition to misreading of the 8-OH-dG residue itself, pyrimidines next to the 8-OH-dG residue (G has not yet been tested) were also misread.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.