Distinct mechanisms of cis-syn thymine dimer bypass by Dpo4 and DNA polymerase eta.
UV-light-induced cyclobutane pyrimidine dimers (CPDs) present a severe block to synthesis by replicative DNA polymerases (Pols), whereas Poleta promotes proficient and error-free replication through CPDs. Although the archael Dpo4, which, like Poleta, belongs to the Y family of DNA Pols, can also replicate through a CPD, it is much less efficient than Poleta. The x-ray crystal structure of Dpo4 complexed with either the 3'-thymine (T) or the 5' T of a cis-syn TT dimer has indicated that, whereas the 3' T of the dimer forms a Watson-Crick base pair with the incoming dideoxy ATP, the 5' T forms a Hoogsteen base pair with the dideoxy ATP in syn conformation. Based upon these observations, a similar mechanism involving Hoogsteen base pairing of the 5' T of the dimer with the incoming A has been proposed for Poleta. Here we examine the mechanisms of CPD bypass by Dpo4 and Poleta using nucleotide analogs that specifically disrupt the Hoogsteen or Watson-Crick base pairing. Our results show that both Dpo4 and Poleta incorporate dATP opposite the 5' T of the CPD via Watson-Crick base pairing and not by Hoogsteen base pairing. Furthermore, opposite the 3' T of the dimer, the two Pols differ strikingly in the mechanisms of dATP incorporation, with Dpo4 incorporating opposite an abasic-like intermediate and Poleta using the normal Watson-Crick base pairing. These observations have important implications for the mechanisms used for the inefficient vs. efficient bypass of CPDs by DNA Pols.
Johnson
Robert E
RE
Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Blocker Medical Research Building, Galveston, TX 77555-1061, USA.
Prakash
Louise
L
Prakash
Satya
S
D020029:Base Pairing; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D011740:Pyrimidine Dimers / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011740:Pyrimidine Dimers; D011994:Recombinant Proteins; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases