The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.
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.
McLenigan
M P
MP
Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2725, USA.
Kulaeva
O I
OI
Ennis
D G
DG
Levine
A S
AS
Woodgate
R
R
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*
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