Inactivating pentapeptide insertions in the fission
yeast replication factor C subunit Rfc2 cluster near
the ATP-binding site and arginine finger motif
Fiona C. Gray
1,2
, Kathryn A. Whitehead
1,
* and Stuart A. MacNeill
1,2,

1 Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK
2 Department of Biology, University of Copenhagen, Denmark
Introduction
The heteropentameric clamp loader replication factor
C (RFC) plays a key role in chromosome replication
in eukaryotic cells. RFC binds to nascent primer–
template junctions and catalyses the loading of the
ring-shaped sliding clamp, proliferating cell nuclear
antigen (PCNA), onto DNA [1,2]. The homotrimeric
PCNA complex encircles the DNA completely, form-
ing a sliding clamp that tethers DNA polymerase d to
the DNA, conferring upon it the processivity necessary
to efficiently replicate the genome. PCNA also inter-
acts with a large number of additional proteins
implicated in DNA replication, DNA repair and DNA
modification such as DNA ligase I, the nucleases Fen1
and XP-G, uracil-N-glycosylase and cytosine-5-methyl-
transferase [3].
The five subunits of the RFC complex are related to
one another but are not interchangeable [1,2]. The
complex comprises a large subunit, Rfc1, and four

properties of a collection of 38 mutant forms of the Rfc2 protein generated
by pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Each
insertion was tested for its ability to support growth in fission yeast
rfc2D cells lacking endogenous Rfc2 protein and the location of each inser-
tion was mapped onto the 3D structure of budding yeast Rfc2. This analy-
sis revealed that the majority of the inactivating mutations mapped in or
adjacent to ATP sites C and D in Rfc2 (arginine finger and P-loop, respec-
tively) or to the five-stranded b sheet at the heart of the Rfc2 protein. By
contrast, nonlethal mutations map predominantly to loop regions or to the
outer surface of the RFC complex, often in highly conserved regions of the
protein. Possible explanations for the effects of the various insertions are
discussed.
Abbreviations
PCNA, proliferating cell nuclear antigen; PSM, pentapeptide-scanning mutagenesis; RFC, replication factor C.
FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4803
smaller subunits, Rfc2–Rfc5. Genetic analysis in yeast
has shown that each of the five subunits is individually
essential for chromosome replication [4]. Three RFC-
like complexes have also been identified in eukaryotic
cells with diverse but poorly understood roles in vari-
ous aspects of checkpoint control, cohesion establish-
ment and genome stability [5]. In these complexes,
Rfc1 is replaced by one of Rad17, Ctf18 or Elg1. Two
additional subunits are also present in the Ctf18–RFC
complex.
Each of the large and small RFC subunits is a mem-
ber of the AAA
+
family of ATPase and ATPase-like
proteins [6–8], and PCNA loading requires multiple

ATP is bound at four sites in RFC (designated ATP
sites A–D) located at the subunit interfaces [1,2]. Each
site is bipartite in nature, comprising elements pro-
vided by adjacent subunits. Thus ATP site A is com-
posed of the ATP-binding P-loop of Rfc1 (also known
as RFC-A) and an arginine residue located in Rfc4
(RFC-B). The side chain of the arginine is referred to
as an arginine finger and the finger protrudes into the
ATP-binding site of the neighbouring subunit. The
exact biochemical roles of the arginine fingers have not
been precisely defined, but may involve sensing ATP
binding in the P-loop and ⁄ or catalysing subsequent
ATP hydrolysis. The fingers are not required for ATP
binding [10].
In this study, we focus on the Rfc2 protein (also
known as RFC-D). Rfc2 binds ATP in site D at the
Rfc2–Rfc5 (RFC-D–RFC-E) interface and contributes
an arginine finger to site C at the Rfc3–Rfc2 (RFC-C–
RFC-D) interface. Biochemical analysis of the proper-
ties of mutant yeast RFC complexes has shown that
the Rfc2 arginine finger at site C is required for the
RFC–ATP–open PCNA complex to bind DNA, lead-
ing to the proposal that the conformational changes
required for RFC to bind the primer–template DNA
require that the Rfc2 arginine finger responds to the
presence of ATP in site C [10]. ADP cannot substitute
for ATP in these reactions.
The Escherichia coli clamp loader, the c-complex,
loads the b-sliding clamp onto DNA and is broadly
analogous to RFC [1,2]. On the basis of analysis of

or around ATP sites C and D (arginine finger and
P-loop respectively), or in the five-strand parallel b
sheet at the core of Rfc2. By contrast, nonlethal
mutations map predominantly to loop regions or to
the outer surface of the RFC complex, often in highly
Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.
4804 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS
conserved regions of the protein. Possible explana-
tions for the effects of the various insertions are
discussed.
Results and Discussion
Pentapeptide-scanning mutagenesis
Pentapeptide-scanning mutagenesis (PSM) is a rapid
method for the random insertion of variable five
amino acid sequences into a target protein [14]. Here
the system based on transposon Tn4330 was used [13].
Tn4430 contains cleavage sites for the restriction
enzyme KpnI only 5 bp from its termini and duplicates
five nucleotides of target site DNA during transposi-
tion. By allowing Tn4430 to insert into a target DNA,
then digesting the target with KpnI and re-ligating, the
bulk of the transposon is deleted, leaving behind only
15 bp of sequence derived from the ends of the trans-
poson and the target site duplication. Should Tn4430
insertion occur within an ORF, the 15-bp insertion will
generally result in the encoded protein acquiring a five
amino acid (pentapeptide) insertion. Insertion can also
result in the generation of an inframe stop codon, but
owing to the sequence constraints imposed by the
sequence of the transposon ends, this is a relatively

K19 I 67–68 Ser-ArgGlyThrProSer-Thr ))
K9 I 82–83 Met-GlyTyrProLeuMet-Lys K25 + +
K11 I 94–95 Glu-GlyValProHisGlu-Arg + +
K20 I 99–100 Ile-ArgGlyThrProIle-Ile + +
F46 I 124–125 Phe-GlyValProLeuPhe-Lys + +
F49 I 124–125 Phe-ArgGlyThrProPhe-Lys + +
F45 I 146–147 Thr-ArgGlyThrProThr-Met ))
K13 I 149 Ser-Ter ))
F37 I 157–158 Cys-LeuGlyTyrProCys-Leu F38 ))
F39 I 170–171 Leu-ArgGlyThrProLeu-Ser ))
K5 I 171–172 Ser-GlyValProLeuSer-Ser ))
F42 I 174–175 Cys-ArgGlyThrProCys-Ser ))
K26 II 183–184 Asp-ArgGlyThrProAsp-Asn K28 + +
K1 II 195–196 Ala-GlyValProLeuAla-Ala ) ++
F41 III 242–243 Val-GlyValProArgVal-Glu ) +
K35 III 251–252 Tyr-ArgGlyThrProTyr-Asn + +
K15 III 254–255 Ile-ArgGlyThrProIle-Arg + +
F44 III 258–259 Leu-GlyValProLeuLeu-Asp + +
F47 III 305 Lys-GYPSKVQNIHETF-Ter F48, F50 + +
K8 III 331–332 Asp-GlyValProLeuAsp-Leu ) +
F. C. Gray
et al. Mutagenesis of the RFC small subunit Rfc2
FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4805
origins, corresponded to only 31 different alleles (inser-
tions indicated in bold type in Table 1).
In addition to these insertions, generated by straight-
forward Tn4430 transposition and excision, seven
more alleles were constructed by further manipulation
of the original set of mutants. These fell into two clas-
ses. Three alleles were constructed bearing short

mutant proteins in fission yeast, each mutant allele
was cloned 3¢ to the thiamine-repressible nmt1 pro-
moter in the expression vector pREP3XH6 [16] and
Table 2. Extended insertions: location and inserted sequences.
Insertion number Domain Location Inserted sequence Low-level expression High-level expression
K11a I 94–95 Glu-GlyValProProGlyLeu
ValProProGlyLeuValPro
ThrProGlyValProProGly
LeuValPheHisGlu-Arg
) +
K26a II 183–184 Asp-ArgGlyThrProGlyVal
GlyThrProAsp-Asn
++
K26b II 183–184 Asp-ArgGlyThrProGlyGly
ValGlyProGlyValGlyThr
ProAsp-Asn
++
K1a II 195–196 Ala-GlyValProProValGly
LeuGlyProGlyLeuValPro
LeuAla-Ala
++
Fig. 1. Location of pentapeptide insertions in Rfc2 protein. Schematic representation of the fission yeast Rfc2 protein showing the location
of the pentapeptide insertion mutants generated in this study. Light grey box: domain I (amino acids 1–181). White box: domain II (amino
acids 182–246). Dark grey box: domain III (amino acids 247–340). Open circles: functional proteins. Grey filled circles: partly functional
proteins. Black circles: non-functional proteins. See text, Table 1 and Fig. 2 for further details.
Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.
4806 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS
each plasmid transformed individually into an S. pom-
be rfc2
+

protein.
Mapping the insertions onto the structure of
Rfc2: insertions into the N-terminal AAA
+
domain
The 3D structure of the Rfc2 protein comprises three
separate domains. Domains I and II together form the
AAA
+
ATPase module, whereas domain III forms
part of the RFC circular collar and is unique to
clamps loaders [9]. Based on alignment with the bud-
ding yeast Rfc2 protein, the N-terminal AAA
+
domain of fission yeast Rfc2 encompasses amino acid
residues 1–181 (Fig. 1). In the Rfc2 crystal structure
(PDB entry 1SXJ) this domain comprises 11 a-helical
segments and 5 b strands [9]. Here the helices are des-
ignated a1–a3, a3¢ and a4–a10 and the strands b1–b5
(Fig. 2A). Note that in PDB entry 1SXJ, the a3–a3¢
segment is designated as a single a helix (helix 69)
despite there being clear structural discontinuity at res-
idues 84–85. For this reason, residues 70–89 are
denoted here as two separate a helices, a3 and a3 ¢
(Fig. 2A).
Twenty-three insertions were located in this domain
of the protein, the most N-terminal (K10) being
located between residues 6 and 7, and the most C-ter-
minal (F42) between residues 174 and 175. The aver-
age distance between the insertions is eight residues,

disrupt the a10 helix, whereas insertion F45 disrupts
a8. These insertions are likely to affect the positioning
of the arginine finger in site D, thereby blocking DNA
binding by RFC–PCNA.
Of the remaining three inactivating mutations, one
(insertion F37) maps to b-strand b4 in the five-strand
parallel b sheet that comprises the core of domain I
(Figs 2A and 3A). The b4 strand is the central strand
in the sheet [9]; it is likely that disruption of this strand
by pentapeptide insertion will affect the entire sheet
(Fig. 3B). The final two inactivating mutations in
domain I (K13, K14) cause premature termination of
the Rfc2 polypeptide chain.
The remaining 13 insertions in domain I failed to dis-
rupt Rfc2 function. Given that all the pentapeptide
insertion sequences include a proline residue that might
be expected to have a significant effect on the secondary
structure in the vicinity of the insertion, it is perhaps sur-
prising that only 8 of the 29 pentapeptide insertions
investigated in this study (< 30% of the total) abolished
Rfc2 function altogether (black circles in Fig. 1; these
figures exclude the two insertions that resulted in prema-
F. C. Gray et al. Mutagenesis of the RFC small subunit Rfc2
FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4807
A
Fig. 2. Location of insertion sites in conserved regions. Location of insertions in N-terminal AAA
+
domain (domain I, shown in A), central
domain (domain II, B) and C-terminal collar domain (domain III, C) of fission yeast Rfc2. The aligned sequences of 10 Rfc2 proteins from
diverse eukaryotic species are shown, corresponding to S. pombe Rfc2 residues 1–181 (domain I), 182–241 (domain II) and 242–340 (domain

individual arginines in each subunit were not tested
[12].
Our results may imply that the a4 arginines R95
and R101 are not absolutely required for in vivo DNA
binding by Rfc2 or indeed that DNA binding by Rfc2
is nonessential for RFC complex function. Further
biochemical and genetic analysis is required to resolve
this issue. However, as a starting point, we used site-
directed mutagenesis to construct seven new single,
double and triple arginine-to-alanine mutations in fis-
sion yeast Rfc2 (designated Rfc2–S1 to Rfc2–S7), at
R95 and R101 in the a4 helix and at R165, the third
residue previously implicated in DNA binding [12].
Each mutant allele (Table 3) was expressed from the
nmt1 promoter in haploid rfc2D cells exactly as
described above for the pentapeptide insertion
mutants. The results of this are summarized in
Table 3. All seven mutant proteins, including Rfc2–S7
with triple K95A, K101A and K165A substitutions,
were able to rescue for loss of Rfc2 function when
expressed at low level (nmt1 promoter repressed by the
B
C
Fig. 2. Continued.
F. C. Gray et al. Mutagenesis of the RFC small subunit Rfc2
FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4809
presence of thiamine in the growth medium, see
above). Clearly, if DNA binding by Rfc2 does play an
essential role in vivo, then the three arginines are not
required for these interactions to occur. Interestingly,

ever, suggesting that the latter possibility is unlikely.
The third deletion mutation tested here (K23DK14)
deletes much of the a2–b1 loop region and the b1
strand and was not viable. Presumably deleting b1 dis-
rupts the structure of the b sheet at the core of domain
I (Fig. 3). As noted above, insertion F37 in the b4
Table 3. Arginine-to-alanine DNA-binding mutants.
Allele Domain Amino acid changes
Low-level
expression
High-level
expression
S1 I R95A + +
S2 I R101A + +
S3 I R165A + +
S4 I R95A-R101A + +
S5 I R95A-R165A + +
S6 I R101A-R165A + +
S7 I R95A-R101A-R165A + +
A
B
Fig. 3. Mapping of insertion sites onto
budding yeast Rfc2 protein structure. (A) 3D
structure of the budding yeast Rfc2 protein
bound to ATP-cS [9]. The locations of the
eight inactivating insertion mutations are
indicated by the blue circles. The locations
of the three arginines implicated in DNA
binding are shown as R95, R101 and R165
(fission yeast numbering). The coordinates

nmt1 promoter, suggesting that the Rfc2–F41 protein
is functionally impaired (Fig. 1). Helix a11 lies on
the outer surface of the RFC complex. Expanding
the K1 and K26 insertions by addition of sequences
encoding a additional 5 or 10 amino acids (see
Table 2 for details) did not disrupt Rfc2 function
either.
Mapping the insertions onto the structure of
Rfc2: insertions into the C-terminal collar domain
The C-terminal domain (domain III) of Rfc2 encom-
passes amino acid residues 247–340 (Fig. 2C) and com-
prises six a helices (a16–a20). These domains form a
right-handed spiral collar structure from which the
AAA
+
domains I and II appear to hang [9]. Five
insertions map within domain III (Fig. 2C). Insertions
K35, K15 and F44 map within a16, which is located
on the outer surface of the RFC complex (Fig. 3A),
whereas insertion K8 maps centrally within a20
located at the Rfc2–Rfc5 interface. None of these
mutations abolishes Rfc2 function but, as with Rfc2–
F41 described above, Rfc2–K8 was required to be
overexpressed in order to rescue rfc2D cells, implying
that the K8 insertion causes a degree of functional
impairment (Fig. 1).
The insertion in F47 is located in a19 (Fig. 2C), but
this mutation is unusual in that Tn4430 transposition
and subsequent restriction enzyme cleavage and
re-ligation left behind a 16 bp, rather than a 15 bp,

regions play in RFC function.
Experimental procedures
Bacterial and yeast strains and media
E.coli DH5a (Stratagene, La Jolla, CA, USA) was used for
routine cloning steps and FH1046 and DS941 for pentapep-
tide mutagenesis [13]. E. coli was cultured on LB medium.
S. pombe rfc2
+
⁄ rfc2::ura4
+
leu1-32 ⁄ leu1-32 ura4-D18 ⁄ ura4-
D18 ade6-M210 ⁄ ade6-M216 h
)
⁄ h
+
[15] was used for func-
tional testing of rfc2 mutations. S. pombe was cultured on
YE, EMM or ME media [20] as required, and transformed
by electroporation [21].
Pentapeptide mutagenesis
To mutagenise rfc2
+
using the pentapeptide insertion
method, an rfc2
+
cDNA was first amplified by PCR from an
S. pombe cDNA library using oligonucleotides SPRFC2–
5BAM (oligo sequence with BamHI site in lower case and
rfc2
+

(rfc2-K1 to rfc2-K36).
A second mutagenesis was carried out using pBR322–
Rfc2–SB as the target plasmid. This plasmid was created by
cloning the SalI–BamHI fragment carrying the 600 bp
3¢ region of the rfc2
+
ORF from pBR322-Rfc2 into
pBR322. pBR322–Rfc2–SB was then subjected to pentapep-
tide mutagenesis as above. Twelve independent transposon-
containing isolated were identified with insertions in the
SalI–BamHI region; sequence analysis showed that these
represented only nine different insertions. These alleles were
designated rfc2-F37 to rfc2-F49.
Generating deletion and insertion alleles
Deletions alleles were constructed by digesting pREP3XH6–
Rfc2–K plasmids with KpnI (a second KpnI site is located in
the LEU2 gene) and ligating together complementary pieces.
Three alleles were constructed in this way: rfc2-K33 ⁄ K27,
rfc2-K33 ⁄ K22 and rfc2-K23 ⁄ K14. Four insertion alleles were
constructed by digesting the relevant pREP3XH6–Rfc2–K
plasmids with KpnI and ligating in oligonucleotide duplexes
constructed by annealing together the following complemen-
tary pairs of oligonucleotides, either SPRFC2-F1 (5¢-CCC
CGGGGTTGGTAC-3¢) and SPRFC2-F2 (5¢-CAACCC
CGGGGGATG-3¢) to produce a five amino acid extension
(insertion K26a) or SPRFC2-F3 (5¢-CCCCGGTGGGGT
TGGGCCCGGGGTTGGTAC-3¢) and SPRFC2-F4 (5¢-CA
ACCCCGGGCCCAACCCCACCGGGGGTAC-3¢) to gen-
erate a 10 amino acid extension (insertions K1a and K26b).
Insertion K11a resulted from the fortuitous ligation of four

+
[15] by electroporation [21] and transformants
obtained on EMM medium. Individual colonies were then
patched overnight at 32 °C on ME medium to induce spor-
ulation, before being treated overnight with helicase to
break down the asci walls and eliminate vegetative cells.
Spores were then washed with water before being plated
on EMM plates supplemented with adenine (EMM + A),
uracil and adenine (EMM + AU), adenine and thiamine
(EMM + AT) and adenine, uracil and 5 lm thiamine
(EMM + AUT) at 23, 32 and 36.5 °C. Leucine was
omitted from all plates to facilitate selection of pREP3X
plasmids which carry the LEU2 selectable marker. Adenine
is required to permit the growth of haploid cells either the
ade6-M210 or ade6-M216 alleles. The addition of uracil
permits growth of rfc2
+
haploids; in the absence of uracil,
only rfc2::ura4
+
haploids expressing functional Rfc2
proteins can grow. The presence of 5 lm thiamine represses
the nmt1 promoter in pREP3X, reducing rfc2 expression by
a factor of 80–100 compared with cells grown on EMM
without thiamine.
Acknowledgements
We would like to thank Dr Finbarr Hayes (University
of Manchester, UK) for supplying the strains necessary
for PSM. This research was funded by a Wellcome
Trust Senior Fellowship in Basic Biomedical Research.

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