(B) Denaturing urea-PAGE of a 3′ overhang containing a single Okazaki fragment substrate (oJR339, oJR343) treated with NaOH, RNase HII, and RNase HIII over 0.1 to 10 min with 1 mM MgCl 2 and 10 μM MnCl 2. (A) SDS-PAGE of 2 μg purified SSB, RNase HIII, and RNase HII stained with Coomassie brilliant blue. RNase HII and HIII process Okazaki fragments in vitro. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.īacillus subtilis DNA polymerase I DNA repair DNA replication Okazaki fragment RNA-DNA hybrid RNase H YpcP.Ĭopyright © 2019 American Society for Microbiology. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitro IMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. RNA-DNA hybrids are common in chromosomal DNA.
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