The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa

Abstract

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.

Bloom’s syndrome: Why not premature aging?

“…Similar to BLM, WRN is recruited at stalled replication forks, and it is able to catalyze in vitro fork regression as well as conversion of regressed HJs to functional forks (Constantinou et al, 2000; Machwe et al, 2011). Additionally, WRN is able to stimulate the bypass of DNA damage by translesion polymerases, which are specialized polymerases that are able to replicate across damaged DNA, a function that BLM has not been reported to have (Kamath-Loeb et al, 2007; Maddukuri et al, 2014; Maddukuri et al, 2012; Phillips and Sale, 2010). …”

RecQ and Fe–S helicases have unique roles in DNA metabolism dictated by their unwinding directionality, substrate specificity, and protein interactions

“…WRN interacts with several DNA pols (pol δ [163–165] and translesion DNA pols [12,166]) to stimulate DNA synthesis and improve fidelity via WRN’s 3′–5′exonuclease activity. WRN is recruited to stalled replication forks to aid in fork recovery and restart [126,167–172].…”

Translesion Synthesis DNA Polymerase Kappa Is Indispensable for DNA Repair Synthesis in Cisplatin Exposed Dorsal Root Ganglion Neurons

“…Although some studies showed that elevated Pol κ promotes genomic instability [5], other studies showed that Pol κ protects cells from mutagens [10,11] and that Pol κ deficiency may exacerbate mutagenesis [12,13]. Interestingly, studies also demonstrated that Pol κ participates in processing of different types of oxidative lesions, including strand breaks [14], bypass of 8-oxo-dG [15] and abasic sites [16,8], which are base excision repair (BER) substrates, as well as processing of bulky lesions that are substrates of the nucleotide excision repair (NER) pathway [17,18]. This suggests that in neurons, Pol κ might have a role in repair of diverse lesions, including cisplatin:DNA crosslinks, which are typical NER substrates [19] and in fact, cells lacking Pol κ are partially defective in interstrand crosslink removal [20,21].…”