Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase 喂 Involved in Translesion DNA Synthesis

详细信息    查看全文

• 作者：Jinsook Kim ; Insil Song ; Ara Jo ; Joo-Ho Shin ; Hana Cho ; Robert L. Eoff ; F. Peter Guengerich ; Jeong-Yun Choi
• 刊名：Chemical Research in Toxicology
• 出版年：2014
• 出版时间：October 20, 2014
• 年：2014
• 卷：27
• 期：10
• 页码：1837-1852
• 全文大小：541K
• ISSN：1520-5010

文摘

DNA polymerase (pol) 喂 is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol 喂 can bypass various DNA lesions, e.g., N²-ethyl(Et)G, O⁶-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol 喂 on its TLS properties, using the recombinant pol 喂 (residues 1鈥?45) proteins and DNA templates containing a G, N²-EtG, O⁶-MeG, 8-oxoG, or abasic site. The 螖1鈥?5 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg²⁺ (but not with Mn²⁺), coinciding with its steady-state kinetic data showing a 鈭?0-fold increase in k_cat/K_m for nucleotide incorporation opposite templates (only with Mg²⁺). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in k_cat/K_m for nucleotide incorporation opposite templates either with Mg²⁺ or Mn²⁺, except for that opposite N²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). The 螖1鈥?5 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg²⁺), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the 螖1鈥?5 variant, was 鈭?-fold stronger with 0.15 mM Mn²⁺ than with Mg²⁺. The results indicate that the R96G variation severely impairs most of the Mg²⁺- and Mn²⁺-dependent TLS abilities of pol 喂, whereas the 螖1鈥?5 variation selectively and substantially enhances the Mg²⁺-dependent TLS capability of pol 喂, emphasizing the potential translational importance of these pol 喂 genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens.