DnaB Helicase Is Unable to Dissociate RNA-DNA Hybrids

Abstract

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.The difficulty to generate palindromes involving ColE1 origins is a well known paradox (1, 2). Indeed, co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogens broad host range plasmid pSM19035 is one of the few exceptions (3, 4). pPI21, an Escherichia coli plasmid derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. It was recently shown that progression of the replication fork initiated at either of the two potential origins of pPI21 is transiently stalled at the other inversely oriented origin (5). This pausing leads to the accumulation of a specific replication intermediate (RI) 1 containing an internal bubble that spans the distance between both origins. The accumulated RI exists as a series of stereoisomers that may have one or more knots with a different number of nodes within the replicated portion (5). Whether this peculiar replication behavior is specific for S. pyogens pSM19035-derived plasmids or a general feature for all plasmids containing two inversely oriented ColE1 origins is still to be shown. Moreover, nothing is known about the mechanism responsible for the transient pausing of replication forks as they encounter another silent and inversely oriented ColE1 origin; and the palindromic structure of pPI21 made it difficult to find out whether or not both origins were equally competent to initiate DNA replication.To answer all these questions, we constructed a series of E. coli plasmids containing two unidirectional ColE1 origins in either the same or opposite orientations. Two-dimensional agarose gel electrophoresis (6) was then u...

The Clash of Macromolecular Titans: Replication-Transcription Conflicts in Bacteria

“…Furthermore, loss of RNase HIII (the enzyme that processes R-loops in B. subtilis ) increased R-loop abundance and completely stalled the replisome at the conflict regions, but only when the genes were oriented head-on to replication (Figure 1). It is presumed that R-loop formation at head-on genes is problematic for replication forks because the replicative helicase in prokaryotes moves 5’ to 3’ on the lagging strand and is not capable of unwinding RNA:DNA hybrids, which would form on the lagging strand in head-on conflicts (56). Interestingly, however, this model is likely incorrect given that the eukaryotic replicative helicase functions on the leading strand, yet the replisome is still impeded by R-loops resulting from head-on conflicts (18).…”

Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone

“…The fact that replication almost never extends upstream of O H toward the ribosomal DNA genes suggests that any fork reaching O H stalls at that point. By analogy with E. coli Col E1 (38), this could be due to the inability of the replisome helicase to separate regions of RNA-DNA hybrid formed by the short D-strand primer and which persist in mtDNA after replication. If D-loops do indeed mediate fork arrest of O H -proximal replication forks (Fig.…”

The Replicative Helicases of Bacteria, Archaea, and Eukarya Can Unwind RNA-DNA Hybrid Substrates

“…Can Unwind DNA-RNA Hybrid-It has not yet been determined whether the archaeal or eukaryal MCM proteins are capable of unwinding substrates containing RNA, and only limited studies have been performed with the bacterial DnaB protein (12). Thus, the ability of a replicative helicase from bacteria, archaea, and eukarya to unwind RNA-containing substrate was determined.…”