RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro.

Abstract

Escherichia coli rnh mutants defective in RNase H activity display the features of previously described sdrA (stable DNA replication) and dasF (dnaA suppressor) mutants: (i) sustained DNA replication in the absence of protein synthesis, (ii) lack of requirement for dnaA protein and the origin of replication (oriC), and (iii) sensitivity of growth to a rich medium. Both the sdrA mutants (selected for continued DNA replication in the absence of protein synthesis) and the dasF mutants (selected as dnaA suppressors) are defective in RNase H activity, measured in vitro. Furthermore, a 760-basepair fragment containing the rnh+ structural gene complements the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in vivo is in the initiation of a cycle of DNA replication at oriC dependent on dnaA+. In keeping with these results, RNase H contributes to the specificity of dnaA protein-dependent replication initiated at oriC in a partially purified enzyme system. Replication of the Escherichia coli chromosome starts at a unique site (oriC) and proceeds bidirectionally (1-3). The DNA fragment containing oriC has been cloned and sequenced (4, 5). Subsequent analyses of the structure and in vivo function of the oriC region have disclosed that a 245-base-pair sequence is required and that within this region certain unique sequences surrounded by defined stretches of spacer sequences must be strictly maintained (6, 7).The initiation of replication at oriC is dependent on several gene products, particularly dnaA (for review, see ref. 8). The dnaA protein, purified to near homogeneity (9), is an essential component required at an early stage of the initiation reaction in an in vitro system which replicates oriC plasmids (10, 11) and binds a 9-base-pair sequence which appears four times in oriC. The dnaB and dnaC gene products, essential for ongoing replication, are also required during or shortly after initiation (12, 13). RNA polymerase has also been implicated by the inhibitory effect of rifampicin at this stage of replication (14) and by a dnaA suppressor identified with rpoB, the l3-subunit gene of RNA polymerase (15).The initiation of each new round of replication of the E. coli chromosome requires de novo protein synthesis (14,16,17). The biochemical nature of this requirement for protein synthesis is still unknown. Mutants, termed constitutive stable DNA replication (Sdrc) mutants, have been isolated that maintain DNA synthesis despite the absence of protein synthesis (stable DNA replication) (18,19). One of these mutant alleles (sdrA224) maps at 5 min between metD and proA (unpublished observations). The sdrA224 mutant tolerates transposon insertional inactivation of the dnaA gene or oriC deletion (20). A group of extragenic suppressor mutations (dasF) of dnaAts mutations has been located near proA (21). The dasF mutants exhibit the Sdrc phenotype (unpublished observations). These several results suggest the presence of an alternative initiation pathway, distinc...

Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins

“…RNase Hi is not known to participate in oriC-dependent replication but instead prevents initiation from alternative origins by degrading RNA primers (40). Nonetheless, the unusual structure of the dnaQ-rnhA transcriptional unit, which is highly conserved in Salmonella typhimunium (21) (5,8,12,14,23).…”

“…Loss of RNase H activity allows replication to initiate from origins other than oriC, the normal chromosomal replication origin; thus, defects in rnhA can suppress conditional mutations in dnaA, which encodes the chromosomal initiation factor (40). If (14).…”

“…Although RNase Hi participates in the initiation of replication of ColEl plasmids by maturing the RNA primer (10), it is not known to directly participate in chromosomal replication. However, by degrading RNA primers at alternative origins, RNase Hi does ensure that replication normally initiates exclusively at oriC (40). Both epsilon and RNase Hi have been implicated in mutagenesis and DNA repair; thus, control of the cellular levels of these proteins may be important both to normal replication and to DNA synthesis on damaged templates.…”

RNA Processing and Degradation inBacillus subtilis

“…RNase H. RNase H cleaves RNA in RNA-DNA hybrid molecules endonucleolytically (106). Its primary function in E. coli seems to be to prevent aberrant DNA replication by degrading potential RNA primers of DNA synthesis at sites other than oriC (117,172). E. coli has two genes encoding RNase H enzymes, rnhA and rnhB, encoding RNase HI and RNase HII, respectively (30,97).…”

The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA

“…Under RNase H-defective conditions, a dnaA-and oriCindependent replication system can function in E. coli (23,38). We attempted to clone new replication origin(s) activated in mh mutant E. coli cells.…”