Biomedical subjects

K Tougu

Publications and source records attributed to K Tougu.

3 recordsLinked to original sources

PubMed1996

The extreme C terminus of primase is required for interaction with DnaB at the replication fork.

We have shown previously that a protein-protein interaction between DnaG and DnaB is required to attract the primase to the replication fork. This interaction was mediated by the C-terminal 16-kDa domain (p16) of the primase. A screen was developed that allowed the detection of mutant p16 proteins that did not interact with DnaB. Various mutagenesis protocols were used to localize this interaction domain to the extreme C terminus of the primase. A mutant primase missing only the C-terminal 16 amino acids was isolated and its activities examined. This mutant enzyme was fully active as a primase, but was incapable of interacting with DnaB. Thus, the mutant primase could not support DNA synthesis in either the general priming reaction or during phiX174 complementary strand DNA replication. Alanine cluster mutagenesis and deletion analysis in p16 allowed the further localization of the interaction domain to the extreme C-terminal 8 amino acids in primase.

Alanine↗

PubMed1996

The interaction between helicase and primase sets the replication fork clock.

The synthesis of an Okazaki fragment occurs once every 1-2 s at the Escherichia coli replication fork and requires precise coordination of the enzymatic activities required. We have shown previously that the primase is recruited anew from solution for each cycle of Okazaki fragment synthesis and that association of primase with the replication fork is via a protein-protein interaction with the helicase, DnaB. We describe here mutant primases that have an altered interaction with DnaB and that direct the synthesis of Okazaki fragments of altered length compared to the wild-type. The mutant primases were deficient only in their ability to participate in replication reactions where their entry to the DNA was provided by the initial protein-protein interaction with DnaB. The primer synthesis capacity of these proteins remained unaffected, as was their ability to interact with the DNA polymerase III holoenzyme. Neither replication fork rate nor the efficiency of primer utilization was affected at replication forks programmed by the mutant enzymes. Thus, the interaction between DnaG and DnaB at the replication fork is the primary regulator of the cycle of Okazaki fragment synthesis.

Base Sequence↗

PubMed1994

Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.

Bacterial Proteins↗