Human DNA polymerase κ forms nonproductive complexes with matched primer termini but not with mismatched primer termini

Abstract

Human DNA polymerase kappa (pol ) is a member of the Y family of DNA polymerases that function in translesion synthesis. It synthesizes DNA with moderate fidelity and does not efficiently incorporate nucleotides opposite DNA lesions. Pol has the unusual ability to efficiently extend from mismatched primer termini, and it extends readily from nucleotides inserted by other DNA polymerases opposite a variety of DNA lesions. All of this has suggested that pol functions during the extension step of translesion synthesis. Here, we have carried out pre-steady-state kinetic studies of pol using DNA with matched and mismatched primer termini. Interestingly, we find that mismatches present only a modest kinetic barrier to nucleotide incorporation by pol . Moreover, and quite surprisingly, active-site titrations revealed that the concentration of active pol is very low with matched DNA, and from DNA trapping experiments we determined that this was due to the formation of nonproductive protein⅐DNA complexes. In marked contrast, we found that the concentration of active pol was six-fold greater with mismatched DNA than with matched DNA. Thus, pol forms nonproductive complexes with matched but not with mismatched DNA. From these observations, we conclude that pol has evolved to specifically function on DNA substrates with aberrant primer-terminal base pairs, such as the ones it would encounter during the extension step of translesion synthesis.DNA damage ͉ DNA repair ͉ DNA replication ͉ kinetics ͉ mutagenesis M embers of the Y family of DNA polymerases (including pols , , , and Rev1) promote translesion synthesis (1-3). These enzymes use diverse mechanisms for incorporating nucleotides opposite damaged template residues. Pol , for instance, catalyzes the efficient and accurate replication through cyclobutane pyrimidine dimers (4-6) and 8-oxoguanine lesions (7), and the lack of functional pol in humans causes the variant form of xeroderma pigmentosum (8, 9). Structural studies have suggested that the active site of pol is unusually large and can accommodate both the bases of a pyrimidine dimer (10), and from biochemical studies, it has been inferred that pol uses the intrinsic Watson-Crick base pairing ability of the thymine-thymine dimer and of 8-oxoguanine to incorporate the correct incoming nucleotide opposite these lesions (11)(12)(13)(14).Unlike pol and every other DNA polymerase examined so far, neither pol nor the Rev1 protein utilizes Watson-Crick base pairing to incorporate nucleotides. Pol catalyzes nucleotide incorporation opposite a variety of DNA lesions, including abasic sites (15, 16), the 3Ј T of (6-4) photoproducts (15-17), and the N 2 -adducted G residues (18,19). The structures of pol in ternary complexes with DNA and dNTPs show that this enzyme uses Hoogsteen base pairing between the template residue and the incoming nucleotide (20,21). This allows pol to replicate through template lesions not capable of forming Watson-Crick base pairs or template lesions containing substantial minor groove modifications (1...

Three Residues of the Interdomain Linker Determine the Conformation and Single-base Deletion Fidelity of Y-family Translesion Polymerases

“…The ability to extend from a C-C mismatched primer terminus, which is enhanced by the Dpo4 linker sequences, is comparable with what has been reported for human polymerase (3,29). For pol , extension from the mismatch involves realigning the slipped DNA strands (29), and we presume that this is the case here.…”

“…Translesion synthesis (TLS) 3 polymerases have the ability to effectively bypass sites of DNA damage, an activity that is crucial to cell survival (1). Functionally, TLS polymerases lack a 3Ј to 5Ј proofreading activity, and structurally, they lack the extensive protein-DNA contacts found in their replicative counterparts that ensure a tightly constrained active site.…”

“…Even when replicating undamaged DNA, the Y-family polymerases each display unique mutagenic signatures (see, for example, Refs. [3][4][5][6][7][8].…”

DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

“…[10][11][12] In line with this, Pol κ and Pol ζ act as extender polymerases after many insults. 9,13,14 Intriguingly, the catalytic activity of REV1 might not be relevant for efficient TLS and, instead, its unique ability to recruit other TLS polymerases to damaged DNA was shown to be central for lesion bypass. 8,15 The recruitment of Pol η to damaged DNA has been elegantly linked to PCNA ubiquitination.…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…34 Curiously, both DinB and pol k proficiently extend from a variety of lesions and mismatched primer ends in addition to their insertion specificities. 8,11,[35][36][37][38][39] It has been suggested, at least for pol k, that this property reflects a separate role in the extension step of TLS. 35 …”