RNase HIII Is Important for Okazaki Fragment Processing in .

RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment using purified recombinant enzymes from We showed that RNase HII and HIII are capable of incision on Okazaki fragments and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation 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. 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 All cells are required to resolve the different types of RNA-DNA hybrids that form When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. 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 .