NIH Research Festival
Ribonucleotides are frequently mis-incorporated into the genome during cellular DNA replication, resulting in genomic instability and mutations. Although DNA repair mechanisms that replace mono-ribonucleotides are known to involve nucleolytic incision by RNaseH1/2 or Topoisomerase I, it is unclear if other DNA binding proteins affect recognition/incision of the lesion. This prompted us to assess the effects of a site- and strand-specific uridylate (rU) or cytidylate (rC) harbored within forked duplex DNA substrate on unwinding catalyzed by disease relevant helicases implicated in replication or DNA repair. The ring-like replicative DNA helicases MCM and Twinkle are tolerant of a rU or rC lesion. In contrast, the repair helicase RECQL1, mutated in the premature aging disorder RECON syndrome, is strongly inhibited by a rU or rC positioned in the helicase-translocating strand. To probe the mechanism further, we tested the clinically relevant and catalytically compromised RECQL1-A459S mutant and found exacerbated inhibition by the translocating strand rU. RECQL1 was sequestered by the rU in the translocating strand of the helicase substrate, suggesting that the helicase is trapped by the ribonucleotide upon encountering it. Moreover, RECQL1 trapped at the translocating rU protects the duplex from both HaeIII endonuclease cleavage at a restriction site adjacent to the ribonucleotide and nucleolytic incision by the ribonucleotide excision repair endoribonuclease RNase H2. These results suggest that a single ribonucleotide can affect genomic transactions of RECQL1 by inducing trapping at the damage site, which may serve to signal DNA repair machinery for localization, or possibly interfere with normal DNA transactions.
Scientific Focus Area: Molecular Biology and Biochemistry
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