LABORATORY OF GENOMIC INTEGRITY

Roger Woodgate, PhD, Chief

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. Recent studies have shown that many of the proteins long implicated in the mutagenic process are, in fact, low-fidelity DNA polymerases that can replicate by moving past damaged DNA in a process termed translesion DNA synthesis (TLS). Humans with defects in one such polymerase, pol eta, are afflicted with xeroderma pigmentosum; they exhibit 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 and included studies on organisms from all three kingdoms of life. In E. coli, the studies centered on polV and its ability to facilitate translesion replication. Scientists in the LGI previously identified, cloned, and characterized a DinB homolog from the archaeon Sulfolobus solfataricus P2, called DNA polymerase IV (Dpo4). In a recent collaborative study, they crystallized the enzyme and solved by X-ray crystallography the structure of ternary complexes of the polymerase together with a cyclobutane pyrimidine dimer and an incoming nucleotide. These structural studies revealed that the active site of the enzyme is sufficiently large to accommodate a covalently linked pyrimidine dimer and thus provided a model for how eukaryotic polymerases, such as human pol eta, can efficiently and accurately bypass a pyrimidine dimer.

Studies with murine DNA polymerase iota revealed that it possesses enzymatic properties similar to human pol iota in that it exhibits a remarkable template-dependent misincorporation spectrum on undamaged DNA in vitro. During attempts to make a targeted disruption of murine pol iota, scientists within the LGI serendipitously discovered that the commonly used 129 strain of mice carries a single nucleotide polymorphism in the Pol iota gene that changes the Serine 27 codon to an amber stop codon and abrogates synthesis of the polymerase. Because pol iota is an extremely error-prone polymerase, it has been hypothesized to participate in somatic hypermutation of immunoglobulin-variable genes. Analysis of somatic hypermutation in the pol iota-deficient 129 mice revealed, however, that the mice exhibit normal levels of mutagenesis and a normal mutation spectrum. Thus, it appears that either pol iota does not participate in somatic hypermutation or its role is nonessential and has been assumed by another low-fidelity DNA polymerase.