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Papers In Press, published online ahead of print May 10, 2002
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331
Corresponding Author: haysj{at}bcc.orst.edu
Mismatch-repair (MMR) systems enhance genomic stability by correcting DNA replication errors. The events in mammalian MMR pathways remain poorly understood. Using Hela-cell nuclear extracts, we analyzed correction of mispairs in circular DNA substrates with single defined nicks, and measured excision in the absence of exogenous dNTPs by annealing specific oligonucleotide probes. In reactions initiated by concomitant temperature shift and addition of ATP or Mg2+ to otherwise-complete mixtures on ice, ATP-initiated excision and final error correction lagged behind Mg2+-initiated reactions, suggesting a very early requirement for ATP, but not its hydrolysis. Subsequent stable commitment (resistance to added excess competitor substrate) began within 30 sec, required hydrolyzable ATP, and plateaued after 60 - 70 sec; this may reflect formation of hydrolysis-dependent translocating and/or preexcision complexes. Excision along shorter nick-mispair paths began 15 sec later than commitment. Both 3'-5' and 5'-3' excision gaps appeared at rates of approximately 0.0055 of final yields per sec, respectively 30 or 2.5 times nonspecific excision rates. The lag between 3' to 5' excision gaps at two different positions yielded an excision progress rate of 5.2 nt/sec. In both substrates, corrected products appeared at fractional rates of 0.0027 of final yield per sec. Aphidicolin, known to inhibit both the DNA-synthesis and 3' to 5' exonuclease activities of pol delta and pol epsilon, reduced appearance of 3' to 5' excision tracts roughly 4-fold at 90 mM, but had no effect on 5' to 3' excision.
J. Biol. Chem, 10.1074/jbc.M200358200
Submitted on January 11, 2002
Revised on May 8, 2002
Accepted on May 9, 2002
Mismatch repair in human nuclear extracts: Time courses and ATP requirements for kinetically distinguishable steps leading to tightly controlled 5' to 3' and aphidicolin-sensitive 3' to 5' mispair-provoked excision
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