title: The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.
abstract: The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.
authors.affiliation: Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2725, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: McLenigan
authors.forename: O I
authors.initials: OI
authors.lastname: Kulaeva
authors.forename: D G
authors.initials: DG
authors.lastname: Ennis
authors.forename: A S
authors.initials: AS
authors.lastname: Levine
authors.forename: R
authors.initials: R
authors.lastname: Woodgate
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017101:Bacteriophage P1 / Q000235:genetics* / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000378:metabolism; D013014:SOS Response, Genetics; D016415:Sequence Alignment; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C400771:HumD protein, Enterobacteria phage P1; D014764:Viral Proteins; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli