Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

Abstract

Nucleotide incorporation and extension opposite N 2 -ethylGua by DNA polymerase was measured and structures of the DNA polymerase -N 2 -ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase opposite N 2 -ethyl-Gua was determined by steady state kinetic analysis with Mg 2؉ or Mn 2؉ as the activating metal. DNA polymerase incorporates dCMP opposite N 2 -ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg 2؉ and with greater efficiencies in the presence of Mn 2؉ . However, the fidelity of nucleotide incorporation by DNA polymerase opposite N 2 -ethyl-Gua and Gua using Mn 2؉ is lower relative to that using Mg 2؉ indicating a metal-dependent effect. DNA polymerase extends from the N 2 -ethyl-Gua:Cyt 3 terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase catalyzed reaction is well suited for N 2 -ethyl-Gua bypass. The structure of DNA polymerase with N 2 -ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase complexed with N 2 -ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.2 is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2Ј-deoxyguanosine-3Ј-monophosphate (1). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2). N 2 -Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2, 3). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4). The formation of N 2 -ethylGua during the reduction of acetaldehyde could cause ethanolrelated cancers (5).The ethyl moiety of N 2 -ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6, 7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8). The blocking of replicative DNA pols by N 2 -ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8). N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA po...

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

“…Formation of a Hoogsteen base pair during nucleotide incorporation is a unique property of pol , allowing it to bypass minor groove DNA adducts efficiently, e.g. the N2 position of G. The N 2 -ethylG lesion, for example, is rotated out of the active site of pol and does not interfere with hydrogen bond formation between the template base and incoming nucleotide (24). In contrast, the major groove O 6 -methylG adduct, when rotated to the syn conformation, remains on the hydrogen bonding face of the template base and influences the hydrogen bond- ing properties of the adduct.…”

“…Interestingly, for minor groove DNA adducts, e.g. at the N2 position of G, the ability of pol to form Hoogsteen base pairs results in high efficiency of correct nucleotide incorporation (24,39 -methylG compared with dCTP. Different hydrogen bonds were formed whether dCTP or dTTP pair with O 6 -methylG in the pol active site (Fig.…”

“…The N3 atom of cytosine must be protonated to function as a hydrogen donor to the N7 and O6 atoms of O 6 -methylG. A positively charged cytosine has been hypothesized to bind in the active site of pol , as a hydrogen atom at the N3 position is needed to form a base pair with the syn G-and N 2 -ethylGtemplate bases (24,25). The geometry of cytosine, bound in the active site of pol , suggests that the N3 atom opposite O 6 -methylG is protonated and donates its hydrogen to the N7 and O6 acceptor atoms of O 6 -methylG in a bifurcated hydrogen bond (Fig.…”

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

“…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.…”

Metals in Biology 2016: Molecular Basis of Selection of Metals by Enzymes

“…However, manganese, cobalt, and calcium can support activity of some DNA polymerases under certain conditions, but the general pattern is that the alternate metals decrease fidelity. However, translesion DNA polymerase prefers manganese over magnesium (14,15). The reasons for metal ion selectivity are structural, and details about the process are becoming available.…”