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PDBsum entry 2p5g
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Transferase/DNA
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PDB id
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2p5g
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References listed in PDB file
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Key reference
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Title
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Caught bending the a-Rule: crystal structures of translesion DNA synthesis with a non-Natural nucleotide.
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Authors
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K.E.Zahn,
H.Belrhali,
S.S.Wallace,
S.Doublié.
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Ref.
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Biochemistry, 2007,
46,
10551-10561.
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PubMed id
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Abstract
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Damage to DNA involving excision of the nucleobase at the N-glycosidic bond
forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired
abasic site during DNA synthesis, most B family polymerases obey the A-rule and
preferentially incorporate dAMP without instruction from the template. In
addition to being potentially mutagenic, abasic sites provide strong blocks to
DNA synthesis. A previous crystal structure of an exonuclease deficient variant
of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43
exo-) illustrated these properties, showing that the polymerase failed to
translocate the DNA following insertion of dAMP opposite an abasic site. We
examine four new structures depicting several steps of translesion DNA synthesis
by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue,
5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is
incorporated more efficiently than dAMP opposite abasic sites. Our structures
indicate that a dipole-induced dipole stacking interaction between the 5-nitro
group and base 3' to the templating lesion explains the enhanced kinetics of
5-NITP. As with dAMP, the DNA fails to translocate following insertion of
5-NIMP, although distortions at the nascent primer terminus contribute less than
previously thought in inducing the stall, given that 5-NIMP preserves relatively
undistorted geometry at the insertion site following phosphoryl transfer. An
open ternary configuration, novel in B family polymerases, reveals an initial
template independent binding of 5-NITP adjacent to the active site of the open
polymerase, suggesting that closure of the fingers domain shuttles the
nucleotide to the active site while testing the substrate against the template.
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