DNA Polymerase η Is an &[Iota]mportant &[Beta]ypass DNA Polymerase in the Repair of DNA Interstrand Cross‐Links
Abstract
DNA interstrand cross‐links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti‐cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanism of ICLs is important for understanding the toxicity of DNA cross‐linking agents and for the development of effective chemotherapies. The repair of ICLs in mammalian cells involves sequential endonucleolytic excision of the lesion from one strand and then the other, and translesion synthesis (TLS) is a critical step for bypassing the unhooked intermediates from the nontemplate strand or for conducting postreplication gap‐filling DNA synthesis. A number of TLS DNA polymerases in humans have diverse bypass specificities and replication fidelities. To illustrate the important role of a certain TLS in ICL repair, we used model ICLs induced by oxidized abasic lesions and those‐derived from O6‐2′‐deoxyguanosine. We investigated the DNA replication‐blocking and mutagenic properties of these ICLs during TLS using primer extension assays and steady‐state kinetic analysis of single nucleotide incorporation prior to, opposite and past the cross‐linked template nucleotide. Liquid chromatography‐mass spectrometry‐based oligonucleotide sequencing was also used to determine the relative yield of the mutagenic bypass products. Our results demonstrated that among the several human TLS DNA polymerases tested, pol η exhibits the relatively high bypass activity; however, a significant portion of bypass products is mutagenic and contains substitution or deletion mutations. The importance of pol η in ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol η. Together, this body of work provides in vitro evidence of the important role of DNA polymerase η in ICL repair and underscores the importance of further evaluating of the role of pol η in ICL repair pathways in biological systems.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2017
- Source ID
- 10.1096/fasebj.31.1_supplement.906.11
Entities
People
- Linlin Zhao
Organizations
- Army Research Office
- Central Michigan University