DNA Replication, Repair, and Mutagenesis In Eukaryotic

Child Health And Human Development

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Abstract

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into damaged DNA. It is now known 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, 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 collaborative study, researchers crystallized the enzyme and solved by X-ray crystallography the structure of ternary complexes of the polymerase together with an abasic site and a Benzo[a]pyrene DNA adduct. These structural studies revealed that the active site of the enzyme is large and readily accommodates lesions that normally block high fidelity replicases. The structural studies also demonstrated that the so-called ?little finger? (LF) domain of the polymerase undergoes considerable movement during TLS. The critical role of the LF domain in TLS was further investigated by making chimeras of the closely related Dpo4 and Dbh polymerases that exhibit different biochemical properties. These studies revealed that the biochemical properties of the chimeras were largely dependent upon the origin of the LF domain. Indeed, if it was from Dpo4, the chimeric enzyme was Dpo4-like in nature, and if it was from Dbh, the chimera behaved very much like Dbh. Studies on human DNA polymerase iota focused on understanding the interactions between the polymerase and proliferating cell nuclear antigen (PCNA). PCNA normally interacts with the cell?s high fidelity polymerase endowing it with great processivity. Scientists in the LGI discovered that PCNA also stimulates the processivity of pol iota in a template-dependent manner in vitro. Interestingly, mutations in one of the putative PCNA-binding motifs of pol iota or the interdomain connector loop of PCNA, diminish the binding between pol iota and PCNA and concomitantly reduce PCNA-dependent stimulation of pol iota activity in vitro. Furthermore, whilst retaining its capacity to interact with pol eta in vivo, the pol iota mutant failed to accumulate in replication foci after DNA damage. As a consequense of these findings, scientists in the LGI hypothesized that PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and TLS-polymerases to ensure genome integrity.