RNase HIII Is Important for Okazaki Fragment Processing in Bacillus subtilis

Abstract

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 in vivo. 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 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). 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. 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. 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 in vitro 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 in vitro.

RnhP is a plasmid‐borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610

“…To assay for RNase H activity, we used an oligonucleotide labeled with an IR dye at the 5′ end that contained four embedded rNMPs flanked by DNA on either side. This labeled oligonucleotide was annealed to a complementary DNA strand, creating a double‐stranded RNA‐DNA chimeric substrate as previously described (Randall et al ., 2019). We used this approach because prior work showed that bacterial RNase HI, HII, and HIII are all active on this substrate (Ohtani et al ., 1999a; Randall et al ., 2017; Randall et al ., 2019).…”

“…RNA‐DNA hybrids in the form of Okazaki fragments and DNA polymerase errors are similar in that the RNA is covalently linked to DNA through a phosphodiester bond (Randall et al ., 2017; Randall et al ., 2019). Another prevalent RNA‐DNA hybrid forms during transcription when mRNA transcripts are base‐paired with the transcribed DNA strand, displacing the coding strand as ssDNA to form an R‐loop [for review (Schroeder et al ., 2015)].…”

“…Bacterial RNase H enzymes are grouped into two general types based on amino acid sequence similarity: type I, which includes RNase HI, and type II, which includes RNase HII and HIII (Kochiwa et al ., 2007). RNase HI and RNase HIII enzymes act on both ribopatches (four or more embedded rNMPs) and hybrids lacking a covalent RNA‐DNA junction, but are unable to cleave at a single rNMP embedded in DNA (Nowotny et al ., 2007; Randall et al ., 2017; Randall et al ., 2019). Unlike RNase HI and HIII, RNase HII enzymes are adept for cleavage at single embedded rNMPs and ribopatches participating in ribonucleotide excision repair (RER), yet show very poor activity on hybrids that lack a covalent RNA‐DNA junction (Sparks et al ., 2012; Chon et al ., 2013).…”

Bulk and single-molecule analysis of a bacterial DNA2-like helicase–nuclease reveals a single-stranded DNA looping motor

“…Furthermore, the major roles played by DNA2 in eukaryotic organisms are apparently already provided by other enzymes in bacteria. For example, the AddAB- and RecBCD-type helicase–nucleases are responsible for DNA end resection to promote homologous recombination ( 15 ), and Okazaki fragments are processed by RNaseH ( 41 ), although the latter process remains poorly understood in bacteria ( 42 ). Finally, Bad proteins all contain a central conserved domain that is not present in any eukaryotic DNA2 protein ( Supplementary Figures S1-S3 ).…”

Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

“…RecBCD-type helicase-nucleases are responsible for DNA end resection to promote homologous recombination (15), and Okazaki fragments are processed by RNaseH (39), although the latter process remains surprisingly poorly understood in bacteria (40). Finally, Bad proteins all contain a central conserved domain that is not present in any eukaryotic DNA2 protein (Supplementary Figures 1-3).…”