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J Biol Chem, Vol. 274, Issue 23, 15975-15977, June 4, 1999
From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The RAD30 gene of Saccharomyces
cerevisiae encodes a DNA polymerase, Pol UV-induced DNA damage presents a block to the DNA replication
machinery. To maintain the continuity of the DNA during replication, UV
lesions encountered by the replication machinery are circumvented by
both error-free and error-prone means. In the yeast Saccharomyces cerevisiae, genes in the RAD6 epistasis group function
in the replication of DNA-containing lesions generated by UV light and by other DNA damaging agents. Mutations in the RAD6 and
RAD18 genes confer extreme sensitivity to UV light, and
these mutants are defective in postreplicative bypass of UV-damaged DNA
and in UV-induced mutagenesis (1). Rad6, a ubiquitin-conjugating enzyme, exists in vivo in a complex with Rad18, a
DNA-binding protein (2, 3). How Rad6-Rad18
protein-dependent ubiquitination promotes error-free and
mutagenic postreplicative bypass is not known.
Of the genes in the RAD6 epistasis group,
REV1, REV3, and REV7 are required for
mutagenic bypass of UV damage, and yeast lacking any of these genes is
nonmutable by UV light (1). The Rev3 and Rev7 proteins form DNA
polymerase The presence of biochemical activity in a protein does not necessarily
imply the requirement of that activity in the biological function of
the protein. For example, the deoxycytidyl transferase activity of Rev1
seems to have no role in the bypass of UV-damaged DNA templates (5).
The S. cerevisiae Rad3 protein and its human counterpart,
XPD, both possess DNA helicase activity (11, 12), and they are required
for nucleotide excision repair and for RNA polymerase II transcription.
Mutational inactivation of the DNA helicase activity of these proteins,
however, impairs only the repair function and not the transcription
function (13-15). Thus, the DNA helicase activities of Rad3 and XPD
are required for DNA repair but not for transcription. Here, we examine
whether the DNA polymerase activity of Rad30 is necessary for its role
in damage bypass. For this purpose, we altered the aspartate and glutamate residues present in the highly conserved domain of serine, isoleucine, aspartate, and glutamate (SIDE) in Rad30 to alanines. The
resulting Rad30 mutant protein lacks DNA polymerase activity, and this
mutation inactivates the biological function of RAD30. Thus,
the DNA polymerase activity of Rad30 is indispensable for its role in
damage bypass.
Yeast Strains and Plasmids--
Genetic studies were done using
yeast strain EMY74.7 (MATa his3 Mutation of the RAD30 Gene and Plasmid Constructions--
To
generate the rad30 Asp155 Purification of rad30 Ala155-Ala156
Mutant Protein--
Rad30 Ala155-Ala156 mutant
protein was purified from the protease-deficient yeast strain BJ5464 as
described previously for the wild type Rad30 protein (10).
DNA Polymerase Activity--
DNA polymerase activity was assayed
as described previously (10). Reactions (10 µl) containing 25 mM KPO4 (pH 7.0), 5 mM MgCl2, 5 mM dithiothreitol, 100 µg/ml bovine
serum albumin, 10% glycerol, 100 µM each of the four
dNTPs, and 10 nM of 5' 32P-labeled
oligonucleotide primer annealed to an oligonucleotide template were
incubated for 5 min at 30 °C with either 2.5 nM of wild
type GST-Rad30 protein or 10 nM of GST-Rad30
Ala155-Ala156 mutant protein. Substrates S-1,
S-2, S-3, and S-4 have been described previously (10).
UV Sensitivity and UV Mutagenesis--
Yeast strains were grown
to mid-exponential phase in selective medium, washed, sonicated to
disperse cell clumps when necessary, and resuspended in sterile
distilled water to a density of 2 × 108 cells/ml.
Cell suspensions were diluted, spread onto the appropriate medium, and
irradiated at a dose rate of 1 J/m2/s. Plates were
incubated in the dark, and colonies were counted after 3-5 days. For
UV-induced mutagenesis, cells were processed as described above, and
appropriate dilutions were plated on synthetic complete medium for
viability determinations and on synthetic medium lacking arginine but
supplemented with canavanine for determination of
can1r mutation frequencies.
Conserved Domains in Rad30 and Related Proteins--
Rad30 shares
significant homology with the S. cerevisiae Rev1 protein and
the E. coli UmuC and DinB proteins (8, 9). Previous studies
have indicated that Rev1 is a deoxycytidyl transferase that transfers a
dCMP residue to the 3'-end of a DNA primer in a
template-dependent reaction (5) and that Rad30 is a DNA
polymerase that can efficiently replicate DNA containing a
thymine-thymine dimer (10). The E. coli UmuC and UmuD'
proteins promote damage bypass by DNA polymerase III, but the mechanism
of their action remains to be elucidated (17, 18). DinB is required for
the untargeted mutagenesis of unirradiated
Alignment of the amino acid sequences of Rad30 and its related proteins
indicates the presence of five conserved motifs, I-V (Fig.
1). Of particular interest are motif I,
which contains a conserved aspartate residue flanked by conserved
hydrophobic residues on both sides, and motif III, which contains the
highly conserved sequence SIDE. Even though the Rad30 family of
proteins shares no sequence homology with any other known prokaryotic
or eukaryotic DNA polymerases, motif III resembles motif C, which is
common to the entire polymerase family and which also contains an
invariant aspartate residue and another highly conserved acidic residue (20). Motif I of the Rad30 family may be analogous to motif A, which
also is common to the entire polymerase family and which contains an
invariant aspartate residue (20). Mutational studies of these three
conserved acidic residues in motifs A and C of various polymerases have
indicated a crucial role of these acidic residues in catalysis
(20).
The Rad30 Ala155-Ala156 Mutant Protein
Lacks DNA Polymerase Activity--
Because of the high degree of
conservation of the SIDE sequence present in Rad30 at residues 153-156
and because of the similarity of this domain to motif C of other
polymerases, we changed the Asp155 and Glu156
residues of Rad30 to alanines. Similar to the previously reported work
for the wild type Rad30 protein, the mutant rad30 gene was fused in frame downstream of the GST gene expressed in yeast from the
galactose-inducible phosphogylcerate kinase promoter, and the resulting
fusion protein was purified from a protease-deficient yeast strain
harboring the GST-Rad30 mutant plasmid pBJ643. During purification, the
mutant protein displayed the same chromatographic properties as the
wild type protein, and in SDS-polyacrylamide gel electrophoresis, both
proteins exhibit identical electrophoretic mobility (Fig.
2A). With all four DNA
substrates S-1, S-2, S-3, and S-4, in which a 75-nucleotide oligomer
template has been annealed to 40-, 41-, 42-, and 43-nucleotide 5'
32P-labeled oligomer primers, respectively, the wild type
Rad30 protein carries out extensive DNA synthesis in the presence of all four dNTPs (Fig. 2B, lanes 1-4). In striking
contrast to the robust DNA polymerase activity of the wild type Rad30
protein, the Rad30 Ala155-Ala156 mutant protein
shows no DNA polymerase activity (Fig. 2B, lanes 5-8), even when four times as much mutant protein was used as the
wild type protein. In fact, the mutant protein was unable to add even a
single nucleotide onto the primer strand of any of the DNA substrates.
Thus, the rad30 Ala155-Ala156
mutation completely inactivates the DNA polymerase activity of Rad30.
The rad30 Ala155-Ala156 Mutation
Inactivates the Biological Function of Rad30--
Previous genetic
studies have indicated a role of Rad30 in error-free bypass of UV
lesions. The rad5
To determine whether Rad30 DNA polymerase activity was required
for UV damage bypass, the ability of the rad30
Ala155-Ala156 mutation to complement the
rad30
In summary, inactivation of the DNA polymerase activity of Rad30 causes
complete loss of the biological function of this protein, thus
indicating the requirement of this DNA polymerase activity in damage
bypass. Our studies also suggest the possibility that the aspartate 155 and glutamate 156 residues present in the highly conserved sequence
SIDE in Rad30 play an important role in catalysis. It is possible that
these residues are part of the active site of the enzyme and that they
coordinate the binding of divalent metal ions.
. The Rad30 protein
shares homology with the yeast Rev1 and the Escherichia
coli DinB and UmuC proteins. Although these proteins contain
several highly conserved motifs, only Rad30 has been shown to possess a
DNA polymerase activity. To determine whether the DNA polymerase
activity of Rad30 was essential for its biological function, we made a
mutation in the highly conserved SIDE sequence in Rad30, in which the
aspartate and glutamate residues have each been changed to alanine. The
mutant Rad30 protein lacks the DNA polymerase activity, and the mutant
gene does not complement the rad30
mutation. These
findings indicate that DNA polymerase activity is indispensable for the
biological function of RAD30.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, which shows limited ability to bypass a
cis-syn thymine-thymine dimer (4). Rev1 is a deoxycytidyl transferase that can incorporate a dCMP residue opposite an abasic site
(5). The RAD5 gene is required for error-free
postreplicative bypass of UV lesions, and it encodes a
DNA-dependent ATPase (6, 7). The RAD30 gene
affects an alternate pathway of error-free bypass of UV lesions, and
the rad5 rad30 double mutant exhibits a synergistic
increase in UV sensitivity over either single mutant (8). Rad30 shares
homology with the yeast Rev1 protein and with the Escherichia
coli DinB and UmuC proteins (8, 9). We have recently shown that
RAD30 encodes a novel eukaryotic DNA polymerase, named
Pol, which has the unique ability to efficiently replicate a
cis-syn thymine-thymine dimer-containing template, and it
inserts two A residues across from the dimer (10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-100, leu2-3, 112, trp1, ura3-52) and its derivatives. Genomic deletions of
RAD genes were generated by the gene replacement method. To
generate the rad30 mutation, 1.4- and 1.1-kilobase PCR1 products corresponding
to the 5'- and 3'-flanking regions of the RAD30 gene,
respectively, were directionally cloned into pUC19. The URA3
"geneblaster fragment," which contains the yeast URA3 gene flanked by the duplicated Salmonella typhimurium hisG
gene (16), was then inserted in between these PCR products. The
rad30 deletion generating plasmid, pR30.2, when digested
with the restriction enzyme EcoRI, releases a 6.4-kilobase
fragment, which when introduced into yeast deletes nucleotides from
position +42 to position +1800 of the 1896-nucleotide RAD30
open reading frame. To generate the rad5 mutation, yeast
strains were transformed with the rad5 generating plasmid
pBJ22 digested with XbaI as described (6). Deletions were
confirmed by PCR analysis of genomic DNA. Loss of the URA3
gene was selected for by plating strains on medium containing
5-flouro-orotic acid.
Ala,
Glu156 Ala mutation, we first isolated the wild type
RAD30 gene from the yeast genome by gap repair. The wild
type gene was then cloned into pUC19, generating plasmid pBJ579. To
create the DE AA mutation, the MORPH site-specific mutagenesis kit
(5 Prime 3 Prime, Inc., Boulder, CO) was employed using the
mutagenic oligonucleotide
5'-GTCGAAAGGGCGACTATTGCTGCAGTATTTCTTGATTTGGG-3', which contains the
codons for amino acids AA instead of DE at positions 155 and 156, respectively, in RAD30. To increase the yield of
mutant-containing plasmid, pBJ579 was grown in dut ung E. coli in medium containing uracil and was used as template for DNA
synthesis from the mutagenic primer. The resulting DNA was ligated and
introduced into mutS
E. coli strain
BMH 71-18, which cannot repair mismatches and degrades the
uracil-containing template. Plasmid DNA containing the rad30
DE AA mutation was isolated, and the presence of the mutation was
confirmed by sequencing. The DNA fragment containing the
rad30 mutation was then used to replace the wild type
fragment in the pUC19-derived pBJ579, generating plasmid pBJ639. To
overexpress the mutant protein, the rad30
Ala155-Ala156 mutant gene was fused in-frame
with the glutathione S-transferase gene under control of the
galactose-inducible phosphogylcerate kinase promoter, generating
plasmid pBJ643. Plasmids pBJ640 and pBJ646 contain the wild type and
rad30 Ala155-Ala156 mutant genes,
respectively, in the low copy CEN plasmid YCplac111. These constructs
contain approximately 800 nucleotides of 5'-flanking RAD30 sequence.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
phage grown in pre-UV irradiated E. coli cells (19).
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Fig. 1.
Alignment of S. cerevisiae
Rad30, E. coli DinB, S. cerevisiae
Rev1, and E. coli UmuC amino acid
sequences. Identical and highly conserved residues are
highlighted. Amino acid positions are indicated by
numbers in parentheses. The highly conserved
aspartic acid and glutamic acid residues in motif III that were changed
to alanines in Rad30 are indicated by asterisks. Regions of
homology are indicated by roman numerals I-V and are
described in the text.
View larger version (18K):
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Fig. 2.
Analysis of the Rad30
Ala155-Ala156 mutant protein.
A, purified Rad30 mutant and wild type proteins. Each
protein from the final fractionation step was separated on a 9%
denaturing polyacrylamide gel and stained with Coomassie Blue.
Lane 1, molecular mass standards; lane 2, Rad30
Ala155-Ala156 mutant protein (400 ng);
lane 3, Rad30 wild type protein (300 ng). B, lack
of DNA polymerase activity in the Rad30
Ala155-Ala156 mutant protein. Wild type
GST-Rad30 (2.5 nM) was incubated for 5 min at 30 °C with
DNA substrates (10 nM) S-1, S-2, S-3, and S-4 (lanes
1-4, respectively). The GST-Rad30
Ala155-Ala156 mutant protein (10 nM) was incubated with the DNA substrates (10 nM) S-1, S-2, S-3, and S-4 (lanes 5-8,
respectively) for 5 min at 30 °C. All incubations were carried out
in the presence of all four dNTPs (100 µM each).
nt, nucleotides.
rad30 double mutant exhibits a
synergistic increase in UV sensitivity compared with the
rad5 and rad30 single mutants, and the rate
of UV-induced reversion of the trp1-1 allele is greatly
enhanced in the double mutant over that in either single mutant (8).
These observations have suggested that Rad5 and Rad30 constitute
alternate Rad6-Rad18-dependent pathways for the error-free
bypass of UV-damaged DNA templates.
mutation was examined. The wild type or the mutant
gene was expressed in yeast from the native RAD30 promoter
on a low copy CEN plasmid. As expected, the wild type
RAD30 gene fully complemented the UV sensitivity of the
rad30 mutation and restored UV survival of the
rad30 rad5 strain to the rad5 level
(data not shown). The rad30
Ala155-Ala156 mutation, however, was unable to
complement the UV sensitivity of either the rad30 strain
or the rad5 rad30 strain (Fig.
3A). To determine the effect
of the rad30 Ala155-Ala156 mutation
on UV mutagenesis, we examined the rate of forward mutations at the
CAN1S locus. As shown in Fig. 3B, the
incidence of UV-induced can1r mutations was
higher in the rad30 strain than in the wild type or in
the rad5 strain. Up to the UV dose of 7.5 J/m2, the frequency of can1r
mutations was about the same in the rad5 rad30 strain
as in the rad30 strain, but at 10 J/m2, the
rad5 rad30 strain displays a sharp rise in the
frequency of can1r mutations. Introduction of
the rad30 Ala155-Ala156 mutant gene
in the rad30 strain or in the rad5 rad30
strain had no effect on the incidence of UV-induced
can1r mutations. As expected, the wild type
RAD30 gene lowered the frequency of UV-induced mutations in
the rad30 strain to the wild type level and in the
rad5 rad30 strain to the rad5 level (data not shown).
View larger version (19K):
[in a new window]
Fig. 3.
Lack of complementation of the rad30
mutation by the mutant rad30
Ala155-Ala156 gene. A,
sensitivity to UV irradiation of wild type RAD30 EMY74.7 and
its various isogenic rad30 mutant strains. 
, wild type
strain EMY74.7; 
, rad30 strain YR30-35; 
,
rad5 strain YR5-50; 
, rad5 rad30
double mutant strain YR5-55; 
, rad30 mutant strain
YR30-39 carrying the rad30
Ala155-Ala156 mutation; 
, rad5
rad30 mutant strain YR5-59 carrying the rad30
Ala155-Ala156 mutation. B,
UV-induced CAN1S to can1r
mutations in the wild type strain EMY74.7 and its various isogenic
rad30 mutant strains. Symbols are as described in
A.
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ACKNOWLEDGEMENTS |
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We thank Todd Washington for discussions and Terrance Todd for the generation of the rad30 Ala155-Ala156 mutation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM19261.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Sealy Center for
Molecular Science, University of Texas Medical Branch, 6.104 Medical
Research Bldg., 11th and Mechanic St., Galveston, TX
77555-1061. Tel.: 409-747y8601; Fax: 409-747-8608; E-mail:
lprakash{at}scms.utmb.edu.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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