Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis

DNA damage in the template strand blocks replication by classical DNA polymerases, which are involved in normal DNA replication and repair. In order to overcome these replication blocks, cells employ several non-classical DNA polymerases that are capable of replicating through template DNA lesions in a process called translesion DNA synthesis (1-3). One such enzyme is eukaryotic DNA polymerase eta (pol η), which is a 71-kDa monomeric protein encoded by the RAD30 gene in yeast (4). Pol η functions in the replication of a few types of DNA lesions, including thymine dimers (4-6) and 8-oxoguanines (7,8). Deletion of the RAD30 gene in yeast leads to an increase in ultraviolet (UV) radiation-induced mutagenesis (9-11), and in humans, inactivation of pol η is responsible for the variant form of xeroderma pigmentosum (XPV) (12,13), which results in greater cancer susceptibility. Another non-classical DNA polymerase in eukaryotes is DNA polymerase zeta (pol ζ), which is comprised of a 173-kDa catalytic subunit and a 29-kDa accessory subunit encoded in yeast by the REV3 and REV7 genes, respectively (14,15). Pol ζ functions in the error-prone replication of a wide range of DNA lesions, and disruptions of the REV3 and REV7 genes result in a drastic decrease in the frequency of DNA damage-induced mutations in yeast (16,17). Moreover, expression of anti-sense RNA to pol ζ leads to a reduction in the frequency of UV radiation-induced mutations in human cells (18). A key factor in translesion synthesis is proliferation cell nuclear antigen (PCNA). PCNA, encoded in yeast by the POL30 gene, is a ring-shaped, homotrimeric protein that acts as a sliding clamp for classical DNA polymerases (19,20). Many protein factors involved in DNA replication and repair interact with PCNA via their PCNA interacting peptide (PIP) motifs that bind along the inter-domain connector loop of PCNA (21). Pol η binds to PCNA in this manner, and this interaction is necessary for pol η function in vivo (22,23). Moreover, this interaction stimulates the enzymatic activity of pol η in vitro (22). Pol ζ, although lacking a PIP motif, also interacts with PCNA, and its enzymatic activity is stimulated by PCNA (24). Several PCNA mutant proteins in yeast have been identified that interfere with translesion synthesis in vivo (25-27). One of these is encoded by the pol30-178 allele (formerly called the rev6-1 allele); it encodes a mutant form of PCNA in which Gly-178 is substituted with a serine (25). This amino acid substitution is at the subunit interface of PCNA, and genetic studies have shown that translesion synthesis by both pol η and pol ζ is completely blocked in cells expressing only this mutant form of PCNA (25). All other aspects of DNA replication and repair appear to occur normally in cells expressing this PCNA mutant protein (25). Another PCNA mutant protein that blocks translesion synthesis, but supports normal cell growth is encoded by the pol30-113 allele (26). In this mutant protein, Glu-113 is substituted with a glycine. Interestingly, Glu-113 is also located at the subunit interface of PCNA directly opposite from Gly-178 on the neighboring subunit. Based on the fact that these mutant proteins block translesion synthesis without interfering with normal DNA replication, the structural changes resulting from these amino acid substitutions are likely subtle. To understand the structural and mechanistic basis of the defect in the PCNA G178S mutant protein's ability to support translesion synthesis, we determined the X-ray crystal structure of this mutant protein to a resolution of 2.9 A. We find that the substituted serine side chain forms a new hydrogen bond with the backbone carbonyl of Glu-113 on the adjacent subunit. This contact results in an extended loop on the adjacent subunit (comprising amino acid residues 105-110, which we refer to as loop J) changing its conformation and moving it into an aberrant position that deviates as much as 6.5 A from its position in the wild-type structure. We have examined the biochemical relevance of this structure by carrying out steady state kinetic studies with a model non-classical polymerase, pol η, in the presence of the wild-type and mutant PCNA proteins. We find that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein inhibits incorporation opposite this DNA lesion. These findings suggest that the proper conformation of loop J is essential for translesion DNA synthesis.