Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro

Abstract

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

DNA polymerases in adaptive immunity

“…As Polκ does not seem to play a role in the physiological situation 63,64 , it suggests that this polymerase acts as a back-up when the normal pathway is compromised. Accordingly, the reduction in A/T mutagenesis corresponds well with the lower mutagenicity of Polκ 61,62 . Altogether these data support the proposition that Polη is the sole contributor of A/T mutations during physiological SHM in mice, with other TLS polymerases playing a backup role 65 .…”

“…Half of these mutations consist in A to C and T to G transversion mutations, a pattern that evokes the signature of another TLS enzyme, Polκ 61,62 (Fig.4). As Polκ does not seem to play a role in the physiological situation 63,64 , it suggests that this polymerase acts as a back-up when the normal pathway is compromised.…”

8-(Hydroxymethyl)-3,N4-etheno-C, a Potential Carcinogenic Glycidaldehyde Product, Miscodes In Vitro Using Mammalian DNA Polymerases

“…In a previous report, pol κ showed diverse substrate affinity in its error-free and errorprone bypass of 8-oxo-dG, AP site, AAF-dG and (+)-trans-anti-benzo[α]pyrene-N 2 -dG (22). In the present work, pol κ showed a highly specific insertion preference for both 8-HM-εC and εC, mainly incorporating T opposite the adduct, which would direct a C → A transversion (Fig 4).…”

“…Human pol κ synthesizes undamaged DNA with very low fidelity (21,22). A recent lung tumor study implicates the overexpression of pol κ in tumorigenesis (23), which may account for the accumulation of mutations as the enzyme has very low fidelity.…”

“…Polymerase η (pol η) is coded by the XP-V gene and can bypass cis-syn thymine dimers in an error-free 9/3/01 manner. Polymerase κ (pol κ), the hDINB1 gene product, is a newly identified enzyme with low fidelity and has both error-free and error-prone activities in DNA lesion bypass (20)(21)(22). More recently, O-Wang et al (23) reported the overexpression of pol κ in patients with non-small cell lung cancer.…”