Abstract
The DNA mismatch repair (MMR) system plays a major role in promoting genome stability and suppressing carcinogenesis. In this work, we investigated whether the MMR system is involved in Okazaki fragment maturation. We found that in the yeast Saccharomyces cerevisiae, the MMR system and the flap endonuclease Rad27 act in overlapping pathways that protect the nuclear genome from 1-bp insertions. In addition, we determined that purified yeast and human MutSα proteins recognize 1-nucleotide DNA and RNA flaps. In reconstituted human systems, MutSα, proliferating cell nuclear antigen, and replication factor C activate MutLα endonuclease to remove the flaps. ATPase and endonuclease mutants of MutLα are defective in the flap removal. These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps.
Highlights
The DNA mismatch repair (MMR) system protects humans from cancer
The his7-2 mutation rate for the diploid double mutant msh2⌬/msh2⌬ rad27⌬/ rad27⌬ (13,000 ϫ 10Ϫ8) was increased 36 times relative to the sum of the his7-2 mutation rates for the diploid single mutants msh2⌬/msh2⌬ RAD27/RAD27 and MSH2/MSH2 rad27⌬/ rad27⌬ (Table 3). These findings indicate that there is a functional overlap between the MMR system and Rad27 in haploid and diploid yeast S. cerevisiae. lys2::InsE-A8 is a yeast ϩ1 frameshift reporter [43]. ϩ1 frameshifts that are formed within a 71-bp lys2::InsE-A8 region, including an A8 run, produce Lysϩ cells [43]
Because the MMR systemdependent flap removal (Fig. 5) is likely to occur during CAF1-dependent nucleosome assembly, we studied whether histone H3-H4 deposition by CAF-1 affects the flap-removing activity of the MMR system
Summary
The DNA mismatch repair (MMR) system protects humans from cancer. Results: Combining an MMR system defect (msh2⌬) with rad27⌬ causes a strong synergistic increase in the rate of 1-bp insertions, and a reconstituted MMR system removes 1-nt flaps. MutS␣, proliferating cell nuclear antigen, and replication factor C activate MutL␣ endonuclease to remove the flaps. ATPase and endonuclease mutants of MutL␣ are defective in the flap removal These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps. The reconstituted system bypasses the requirement for exonuclease 1 in the mismatch removal by relying on the strand-displacement activity of DNA polymerase ␦ holoenzyme. Genetic evidence indicates that defective removal of Okazaki fragment flaps causes genome instability [31, 32]. Rad27/FEN1 endonuclease, Dna helicase/nuclease, and the 3Ј 3 5Ј-exonuclease activity of DNA polymerase ␦ remove Okazaki fragment flaps [32,33,34,35,36]. We describe genetic and biochemical experiments that indicate that the MMR system removes 1-nt Okazaki fragment flaps
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