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). These so-called "Y-family" DNA polymerases are found in bacteria, archaea, and eukaryotic cells, indicating that TLS is remarkably conserved throughout evolution. The important role played by these polymerases in mutagenesis and carcinogenesis is typified by human pol eta. Humans with defects in pol eta are afflicted with the Xeroderma Pigmentosum Variant phenotype; they exhibit extreme sensitivity to ultraviolet light and are prone to sunlight-induced skin cancers.
In the past year, experiments aimed at understanding the functions of Y-family polymerases spanned the evolutionary spectrum. For example, studies identified and characterized five novel thermostable Dpo4-like enzymes. The Dpo4-like polymerases were found to be moderately processive and could substitute for Taq polymerase in PCR. By using a blend of Taq and Dpo4-like enzymes, researchers obtained a PCR amplicon from UV-irradiated DNA that could not be amplified 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 the 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 pol 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.
The group investigated in detail the nature of the interactions between RecA and polV. Earlier assumptions held that RecA binds to the damaged DNA template strand being copied by polV. Remarkably, however, the studies revealed that polV-catalyzed translesion synthesis occurs only when RecA nucleoprotein filaments assemble on separate single-stranded (ss)DNA molecules in trans. Furthermore, a 3′-proximal RecA filament end on trans DNA is essential for stimulation and is strengthened by additional polV-RecA interactions occurring elsewhere along a trans nucleoprotein filament. Based on these remarkable observations, the group suggested that, despite the absolute requirement for RecA protein in damage-induced mutagenesis, trans-stimulation of polV by RecA bound to ssDNA reflects a distinctive regulatory mechanism of mutation that resolves the paradox of RecA filaments assembled in cis obstructing translesion DNA synthesis.
Top of Page
