Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12

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Abstract
Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (ΔoriC) and transposon-insertional inactivation of an initiator gene (dnaA:Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein intornh − dnaA: Tn10 and rnh − ΔoriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm−) of the rnh − dnaA: Tn10 recA200 strain was suppressed by the presenceof plasmids (pBR322 derivatives) carrying dnaA +only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and pλasn20) in the absence of required DnaA+ protein nor inhibited dnaA +−dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.