Involvement of Werner syndrome protein in MUTYH-mediated repair of oxidative DNA damage.

ABSTRACT: Reactive oxygen species constantly generated as by-products of cellular metabolism readily attack genomic DNA creating mutagenic lesions such as 7,8-dihydro-8-oxo-guanine (8-oxo-G) that promote aging. 8-oxo-G:A mispairs arising during DNA replication are eliminated by base excision repair initiated by the MutY DNA glycosylase homologue (MUTYH). Here, by using formaldehyde crosslinking in mammalian cell extracts, we demonstrate that the WRN helicase/exonuclease defective in the premature aging disorder Werner syndrome (WS) is recruited to DNA duplex containing an 8-oxo-G:A mispair in a manner dependent on DNA polymerase ? (Pol?) that catalyzes accurate DNA synthesis over 8-oxo-G. Similarly, by immunofluorescence, we show that Pol? is required for accumulation of WRN at sites of 8-oxo-G lesions in human cells. Moreover, we show that nuclear focus formation of WRN and Pol? induced by oxidative stress is dependent on ongoing DNA replication and on the presence of MUTYH. Cell viability assays reveal that depletion of MUTYH suppresses the hypersensitivity of cells lacking WRN and/or Pol? to oxidative stress. Biochemical studies demonstrate that WRN binds to the catalytic domain of Pol? and specifically stimulates DNA gap filling by Pol? over 8-oxo-G followed by strand displacement synthesis. Our results suggest that WRN promotes long-patch DNA repair synthesis by Pol? during MUTYH-initiated repair of 8-oxo-G:A mispairs.

SUBMITTER: Kanagaraj R 

PROVIDER: S-EPMC3458577 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature