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.

摘要: Abstract 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 circular substrates with single defined nicks and measured excision the absence exogenous dNTPs annealing specific oligonucleotide probes. In reactions initiated concomitant temperature shift addition ATP or Mg2+ to otherwise complete mixtures on ice, ATP-initiated final error lagged behind Mg2+-initiated reactions, suggesting a very early requirement for but not its hydrolysis. Subsequent stable commitment (resistance added excess competitor substrate) began within 30 s, required hydrolyzable ATP, plateaued after 60–70 s. This may reflect formation hydrolysis-dependent translocating and/or pre-excision complexes. Excision along shorter nick-mispair paths 15 s later than commitment. Both 3′ 5′ gaps appeared at rates ∼0.0055 yields per second, respectively, 2.5 times nonspecific rates. lag between two different positions yielded an progress rate 5.2 nucleotides/s. both substrates, corrected products fractional 0.0027 yield second. Aphidicolin, known inhibit synthesis exonuclease activities polymerases δ e, reduced appearance tracts roughly 4-fold 90 μm had no effect excision.