Kinetic Analysis of Translesion Synthesis Opposite Bulky N2- and O6-Alkylguanine DNA Adducts by Human DNA Polymerase REV1

Abstract

REV1, a Y family DNA polymerase (pol), is involved in replicative bypass past DNA lesions, so-called translesion DNA synthesis. In addition to a structural role as a scaffold protein, REV1 has been proposed to play a catalytic role as a dCTP transferase in translesion DNA synthesis past abasic and guanine lesions in eukaryotes. To better understand the catalytic function of REV1 in guanine lesion bypass, purified recombinant human REV1 was studied with two series of guanine lesions, N 2 -alkylG adducts (in oligonucleotides) ranging in size from methyl (Me) to CH 2 (6-benzo[a]pyrenyl) (BP) and O 6 -alkylG adducts ranging from Me to 4-oxo-4-(3-pyridyl)butyl (Pob). REV1 readily produced 1-base incorporation opposite G and all G adducts except for O 6 -PobG, which caused almost complete blockage. Steadystate kinetic parameters (k cat /K m ) were similar for insertion of dCTP opposite G and N 2 -G adducts but were severely reduced opposite the O 6 -G adducts. REV1 showed apparent pre-steadystate burst kinetics for dCTP incorporation only opposite N 2 -BPG and little, if any, opposite G, N 2 -benzyl (Bz)G, or O 6 -BzG. The maximal polymerization rate (k pol 0.9 s ؊1 ) opposite N 2 -BPG was almost the same as opposite G, with only slightly decreased binding affinity to dCTP (2.5-fold). REV1 bound N 2 -BPG-adducted DNA 3-fold more tightly than unmodified G-containing DNA. These results and the lack of an elemental effect ((S p )-2-deoxycytidine 5-O-(1-thiotriphosphate)) suggest that the late steps after product formation (possibly product release) become rate-limiting in catalysis opposite N 2 -BPG. We conclude that human REV1, apparently the slowest Y family polymerase, is kinetically highly tolerant to N 2 -adduct at G but not to O 6 -adducts.Cellular DNA is continuously attacked by various endogenous and exogenous agents. Although the resulting lesions can be removed by versatile cellular repair systems, many DNA lesions escape repair and are usually present in replicating DNA. Facing DNA lesions during DNA replication, DNA polymerases often show unusual behavior, such as misinsertion, slippage, and blockage, which can give rise to mutations or cell death (1). Therefore, the characterization of interaction of DNA polymerases with DNA lesions is crucial for understanding the mechanism of mutagenesis in cells in detail (2). Human cells possess at least 15 different DNA polymerases, the physiological functions of most of which are still unclear. Replicative DNA polymerases, such as pol 2 ␣, ␦, and ⑀, are intolerant of DNA distortions caused by many DNA lesions and thus are blocked (3). As a tolerance mechanism to this replication blockade, cells utilize the specialized translesion synthesis (TLS) DNA polymerases, which have a spacious active site to replicate past replication fork-blocking lesions (4). Many of human TLS DNA polymerases belong to the recently discovered Y family, including pol , pol , pol , and REV1 (5). Y family members often have different properties of bypass ability and fidelity opposite various DNA ...

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

“…Replicative DNA polymerases are challenged by N-2 bulk on G even as small as Me or Et. Y-family DNA polymerases efficiently replicate past these forms of damage (42)(43)(44)(45)(46). However, the addition of a second Me group to N 2 -MeG poses a strong block to Y-family DNA polymerases unless they use alternate pairing modes (e.g.…”

“…The presence of an Et group has only a limited effect in slowing the Y-family DNA polymerases (42)(43)(44)(45)(46)(47), and small conformational changes occurring in pol have been shown to accommodate the lesion (47). However, distributing the same amount of overall bulk into two 1-carbon units, i.e.…”

“…With human Y-family DNA polymerases, the rates of dNTP incorporation with the four normal dNTPs are slower, and processivity is lower than with the processive A-and B-family DNA polymerases. However, the presence of alkyl (and aralkyl) moieties at the N-2 atom of G is much less inhibitory to Y-family DNA polymerases (42)(43)(44)(45)(46), and with some DNA polymerases even a bulky (6-benzo[a]pyrenyl)methyl moiety is tolerated reasonably well (43)(44)(45). Dpo4, a prototypic Y-family DNA polymerase from the crenarchaeon Sulfolobus solfataricus, was even less sensitive to the effect of increasing bulk than the human Y-family enzymes (46).…”

“…N 2 ,N 2 -Me 2 G (Fig. 1), has a dramatic effect in slowing incorporation of dCTP by human Y-family pol and (42, 44) but not (human) pol or Rev1 (43,45), the latter two of which use non-"Watson-Crick" pairing in alignments for phosphodiester bond formation (48 -50). In the case of Dpo4 and the bulky lesion N 2 -NaphG, we were able to characterize the crystal structures of two forms and provide evidence for the existence of multiple conformations of the polymerase⅐oligonucleotide⅐dNTP complex (46 ) was from PerkinElmer Life Sciences.…”

“…, and 5% (v/v) glycerol at 37°C (42)(43)(44)(45). All reactions were initiated by mixing dNTP/MgCl 2 (final MgCl 2 concentration of 5 mM) solution to preincubated Dpo4/DNA mixtures.…”