Roger Woodgate, PhD, Chief

Scientists within the Laboratory of Genomic Integrity (LGI) are trying to understand the mechanisms by which mutations are introduced into damaged DNA. Many of the proteins long implicated in the mutagenic process are now known to be low-fidelity DNA polymerases that can replicate past damaged DNA in a process termed translesion DNA synthesis (TLS). In the past year, experiments aimed at understanding the functions of these socalled Y-family DNA polymerases have spanned the evolutionary spectrum and included studies on organisms from all kingdoms of life: bacteria, archaea, and eukaryotes.

As part of ongoing collaborative studies, researchers solved the crystal structures of DNA polymerase IV (Dpo4) from the archaeon Sulfolobus solfataricus, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. Unlike high-fidelity polymerases, Dpo4 lacks an intrinsic 3´-5´ exonuclease to proofread errors. Instead, the crystal structures suggested that pyrophosphorolysis (i.e., the reverse chemical reaction of polymerization) might function as a “proofreading” mechanism, given that it is active only on mispaired substrates and not on the correctly paired “Watson-Crick” bases.

Members of the LGI identified and characterized five novel thermostable Dpo4-like enzymes. The Dpo4-like polymerases were moderately processive and could substitute for Taq polymerase in PCR. By using a blend of Taq and Dpo4-like enzymes, the researchers obtained a PCR amplicon from UV-irradiated DNA that was unamplifiable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCR reactions therefore augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

Studies with human Y-family DNA polymerases iota and eta revealed that they physically interact with ubiquitin. Interestingly, mutant polymerases unable to interact with ubiquitin exhibited significantly lower levels of replication foci in response to DNA damage. Thus, pol eta’s and iota’s ability to bind to ubiquitin is a key step in delivering the TLS polymerases to sites of DNA damage, where they can facilitate lesion bypass.

In further collaborative studies, the LGI demonstrated that, in addition to TLS, pol eta binds to DNA and extends its synthesis from D-loop recombination intermediates in which an invading strand serves as the primer for DNA extension. The observations imply that pol eta may have a dual function at stalled replication forks, namely, the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.