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Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

ABSTRACT: Two new, small, early bacteriophage T4 genes, repEA and repEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which are genuine antisense RNAs for the early transcripts, direct synthesis of virion components. Because the T4 genome contains several origins, and because recombination can bypass a primase requirement for retrograde synthesis, neither defects in a single origin nor primase deficiencies are lethal in T4 (Mosig et al., FEMS Microbiol. Rev. 17:83-98, 1995). Therefore, repEA and repEB were expected and found to be important for T4 DNA replication only when activities of other origins were reduced. To investigate the in vivo roles of the two repE genes, we constructed nonsense mutations in each of them and combined them with the motA mutation sip1 that greatly reduces initiation from other origins. As expected, T4 DNA synthesis and progeny production were severely reduced in the double mutants as compared with the single motA mutant, but early transcription of oriE was reduced neither in the motA nor in the repE mutants. Moreover, residual DNA replication and growth of the double mutants were different at different temperatures, suggesting different functions for repEA and repEB. We surmise that the different structures and protein requirements for functioning of the different origins enhance the flexibility of T4 to adapt to varied growth conditions, and we expect that different origins in other organisms with multiorigin chromosomes might differ in structure and function for similar reasons.

SUBMITTER: Vaiskunaite R

PROVIDER: S-EPMC94188 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

Vaiskunaite R R Miller A A Davenport L L Mosig G G

Journal of bacteriology 19991101 22

Two new, small, early bacteriophage T4 genes, repEA and repEB, located within the origin E (oriE) region of T4 DNA replication, affect functioning of this origin. An important and unusual property of the oriE region is that it is transcribed at early and late periods after infection, but in opposite directions (from complementary DNA strands). The early transcripts are mRNAs for RepEA and RepEB proteins, and they can serve as primers for leading-strand DNA synthesis. The late transcripts, which ...[more]

PMID: 10559179

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Project description:The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.

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Dataset Information

Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

Publications

Two new early bacteriophage T4 genes, repEA and repEB, that are important for DNA replication initiated from origin E.

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