Investigation of the Roles of PCNA and 9-1-1 Clamps in Homologous Recombination in Saccharomyces cerevisiae
Homologous recombination (HR) is a mechanism which is found in all forms of life. It is essential for the maintenance of genome stability. It provides high-fidelity repair of DNA double-strand break (DSB) introduced by ionizing radiation and other agents. It also plays crucial roles in eliminating of interstrand crosslinks from chromosome, preventing the collapse of replication forks, maintaining telomere, as well as ensuring the proper segregation of the chromosome homologues in Meiosis I. (1, 2, 3, 4)
The model of homologous recombination developed from genetic and biochemical studies in Saccharomyces cerevisiae is shown in 1 (reviewed in 5, 6). Briefly, the DSBs are first detected by Mre11-Rad50-Xrs2 (MRX) complex. The broken chromosome ends are then processed by the 5'-3' exonuclease to produce single-stranded DNA (ssDNA) tails. RPA which is a heterotrimeric ssDNA-binding protein coats the 3'-ends of single-stranded DNA (ssDNA) and removes its secondary structure. With the assistance of Rad52, recombinase Rad51 replaces RPA and forms a helical structure on the ssDNA called presynaptic filament. It executes homology search for a partner chromosome and form a nascent D-loop structure by strand invasion. Post invasion DNA synthesis is then carried out by polymerase which has not been well identified ( 1). After post-invasion DNA synthesis, DSB repairing is accomplished by several distinct pathways, such as synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR) and Break induced replication (BIR). In the SDSA pathway, the D loop is unwound and the freed ssDNA strand anneals with the complementary ssDNA strand that is associated with the other DSB end( 1, SSDA pathway). The repair product from SDSA is always non-crossover. Alternatively, the displaced ssDNA in D-loop anneals with ssDNA at the other end of DSB ( 1, canonical DSBR pathway) and produces Holliday junctions. The resolution of HJs by a specialized endonuclease can result in either noncrossover or crossover products. If sequence homology is limited to only one side of the DSB, the cell may take a third pathway- Break induced replication ( 1, BIR pathway), where DNA synthesis continues to the end of invaded chromosome (7).
The identification of G2/M arrest after X-ray irradiation in Saccharomyces cerevisiae brought the concept of DNA damage checkpoints (8). In addition to G2/M transition, the existence of other DNA damage checkpoints have also been demonstrated G1/S transition for instance, which delay cell cycle in response to DNA damage to provide time for DNA repair to occur. In S. cerevisiae, DNA damage checkpoints delay the G1/S transition and block the G2/M transition of the cell cycle (8, 9).
The 9-1-1 clamp (Radl7-Mec3-Ddcl complex of budding yeast) is a sliding clamp that is required for the damage checkpoint. The role of 9-1-1 clamp in cell-cycle control has been extensively investigated (reviewed in 12, 13). In cell cycle arrest, 9-1-1 clamp serves as a component of a damage sensor, which is loaded onto damaged DNA and activates a key protein kinase-Mecl, which in turn initiates complex intracellular signaling, that eventually blocks cell cycle progression. Besides its function in cell cycle control, an early study suggested that the 9-1-1 clamp may also have a role in DNA repair per se (21). More recently an investigation of yeast meiosis (22) showed that the lack of the 9-1-1 clamp function delays meiotic recombination and HO induced DSBs (23). It has also been shown that the 9-1-1 clamp interacts with a repair polymerase Pol ζ, and loss of the clamp function decreases Pol ζ dependent spontaneous mutagenesis (24).
Yeast Proliferating Cell Nuclear Antigen (PCNA), a homotrimeric protein complex, is another sliding clamp which is well known for stimulating DNA polymerase (Pol σ and Pol ε) processivity during DNA replication. PCNA is loaded onto the ssDNA-dsDNA junction that has recessed 3'-end by the RFC clamp loader, a heteropentameric protein complex made up of Rfc1-Rfc2-Rfc3-Rfc4-Rfc5 (. 2). RPA stimulates clamp loading (18). The 9-1-1 clamp is loaded onto DNA by its congnate loader, the Rad24-RFC (Rad24-Rfc2-Rfc4-Rfc3-Rfc5) complex (18, 19). In contrast to PCNA loading, 9-1 -1 clamp is loaded preferentially at the recessed 5'-end at an ssDNA-dsDNA junction (. 2) (18, 20). PCNA is also required for HR (14). Besides Pol δ and Pol ε, PCNA also interacts with Pol η (15, 16), and Pol ζ (17), which are involved in DNA repair.
During DBS repairing by HR, the lost genetic information at the break site is recovered by post - invasion DNA synthesis carried out by DNA polymerases which have not been clearly identified. Characteristics of DNA polymerase involved in post-invasion synthesis determine the length of newly synthesized DNA, which in turn affects the tract of gene conversion (see . 2). Therefore, the DNA polymerase has a crucial role in maintaining the integrity of recombinants. Genetic studies in yeast have not identified a single crucial DNA polymerase in HR process suggesting that multiple DNA polymerases are involved in HR.
Two possible candidates of post invasion DNA synthesis are Pol η (Rad30) (25) and Pol ζ (Rev3-Rev7 complex) (26) which have both been studied extensively as translesion DNA polymerases that synthesize over UV-damaged DNA (reviewed in 27-29). The loss of mouse Pol ζ causes embryonic lethality (30-32). Mouse REV3-/- embryonic fibroblasts show genome instability which is similar to cells lacking HR function. Pol ζ function is also required for HR that is induced by an artificial DSB in mouse embryonic fibroblast cells (34). When Pol η was knocked out in chicken DT40 cells, gene conversion in the pseudo-V region of immunoglobulin gene, as well as HR that is induced by restriction endonuclease I-Scel were compromised (35). Biochemical analyses show that human Pol η can extend DNA from D-loop in vitro more efficiently than Pol δ or Pol ι (Iota) which indicates its possible involvement in post invasion DNA synthesis (36).
Failures of DSB repair in mammals are related to many severe genetic diseases. Post-invasion DNA synthesis is such an important step in HR that is essential for high fidelity of repairing. The identification of DNA polymerase involved in this process has been carried for a long time, but without clear conclusive results. Loss of human Pol η causes a xeroderma pigmentosum variant (XP-V), which causes predisposition to skin cancer (38, 39). Pol ζ is involved in UV-repair in error-prone translesion DNA synthesis (28, 40). DNA polymerase δ is preferentially recruited during homologous recombination to promote heteroduplex DNA extension, which is further stimulated by PCNA loading (41, Maloisel et al.). These polymerases have been suggested to be involved in post-invasion DNA synthesis. However, the molecular mechanism is still unclear. Very little is known about the mechanisms that recruits a specific DNA polymerase to recombination intermediates. Recombinases which direct homology search and strand invasion, may have key roles in the polymerase recruitment, but the results from different research regarding this question are contradictory. One in vitro study shows that Rad51 interacts with Pol η and stimulates its DNA synthesis (38), suggesting that Rad51 recruits Pol η for post-invasion DNA synthesis. However, another in vitro study shows that Rad51 on D-loop inhibits access of polymerase for DNA synthesis, and needs to be removed by Rad54 (41). In vivo observation of post-invasion DNA synthesis during meiosis showed that the DNA synthesis starts when Rad51 is removed from DSB site (42), supporting that removal of Rad5l is required for post-invasion DNA synthesis. This apparent inconsistency need to be clarified.
Another intriguing aspect regarding post invasion DNA synthesis in HR is the function of sliding clamps, especially 9-1-1 clamp, in this process. It has been reported recently that PCNA was able to be loaded onto D-loop structure and stimulate DNA synthesis by Pol δ (41). However, the loading of 9-1-1 clamp onto HR intermediate has never been reported. Since 9-1-1 clamp not only induces damage checkpoint signaling, but also involved in the repair of DSB. It is likely that 9-1-1 clamp is also loaded on the HR intermediate and affects recombination process. The choice of DNA polymerases affects accuracy and extent of post-invasion DNA synthesis which may affect "pathway-choice" of HR together with sliding clamp. This research is dedicated to explore the possibility whether DNA sliding clamps are involved in the D-loop extension step of HR. Although a genetic study indicated that PCNA is involved in HR (14), it remains unclear which biochemical step PCNA is functioning. The 9-1-1 clamp (Radl7-Mec3-Ddcl complex) is also involved in both mitotic and meiotic HR processes (22, 23), but biochemical contribution to HR remains elusive. If we succeed in demonstrating novel activities of Pol η, Pol ζ, PCNA, and 9-1-1 clamp in HR, mechanism of high-fidelity DSB repair becomes clearer. In addition, it will provide insights into the mechanism of meiotic recombination. Results of this project will be applicable for human studies, ultimately for preventing cancer and chromosome disorders caused by meiotic non-disjunction
The following questions will be addressed in this research:
Question-I: Are PCNA and 9-1-1 clamps loaded onto the D-loop by their cognate clamp loaders? 9-1-1 Clamp loading inside the D-loop has not been reported, although D-loop is the place of.post-invasion DNA synthesis. This possibility will be examined extensively.
Question-2: If PCNA or the 9-1-1 clamp are loaded on the D-loop, do they stimulate Pol η or Pol ζ mediated post-invasion DNA synthesis?
Plasmids construction, protein expression and purification. The following recombination proteins are needed for the proposed in vitro assays: ssDNA binding protein RPA, recombinase-Rad51, two DNA polymerases-Pol η (Rad30), and Pol ζ (Rev3 and Rev7 complex), two DNA sliding clamps (PCNA and 9-1-1 clamp), and two clamp loaders (Rad24-RFC and RFC). Among these proteins, Rad51, and RPA have been purified in our previous studies. Rad30 has been purified by lab colleague ( 3. C).
PCNA were cloned into pET21(a) bacterial vector, which fuses these genes with a 6-histidine affinity purification tag. The plasmid was transformed into E. coli, induced by IPTG, and purified to homogeneity ( 3, A).
In the preliminary experiments to overexpress and purify 9-1-1 complex, the subunit genes- RAD17, MEC3 and DDC1, were separately cloned into pET21(a) bacterial expression vector. Three constructs were then separately introduced into E. coli BLR(condon+) strain, and induced by IPTG. Since Rad17 and Ddc1 subunits had low solubility, these subunits were resolubilized under denaturing conditions during purification and reconstituted into heterotrimer in vitro ( 3. B).
Both Rad24-RFC2, 3, 4, 5 (clamp loader of 9-1-1 clamp) and RFC1-5(clamp loader of PCNA) are hetero-pentameric protein complexes which share four common small subunits. The expression strategy of Rad24-RFC2, 3, 4, 5 was to clone them onto three yeast protein expression vectors (RAD24 onto pJN58, RFC2, 3 onto pESCTrp1, RFC4, 5 onto RFCLeu2). All the subunit genes are under the control of galactose inducible bidirectional promoter. Similarly, RFC1 was cloned onto pJN58. Rad24 and RFC1 are expressed as GST-fusion proteins. Rad24-RFC has been overexpressed in yeast and purified to near homogeneity ( 3, E) and ready to test the 9-1-1 clamp loading activities. Purification of Pol ζ and RFC1-5 clamp loader are in progress.
Genomic integration of pESCTrp-RFC2,3 and pESCLeu-RFC4,5 Although Rad24-RFC2-5 complex was produced by the method described above, the yield in only approximately 250µg/100g wet cell. According to replica printing analysis, only about 25% of the cells carry all the three plasmids as a result of plasmid lost during segregation (data not shown). To elevate protein yield, a strategy for stable protein expression in yeast being tested is the integration of pESC-Leu-RFC2/3 and pESC-Trp-RFC4/5 into the wild type yeast strain genome by linear DNA transformation and homologous recombination ( 4). Integrated plasmids in chromosome should be much more stable than episomes in yeast. To construct such yeast strain, yeast replication origins on pESCLeu-RFC2/3 and pESCTrp-RFC4/5 plasmids was first deleted. The modified plasmids are linearlized and then introduced into WT strain TSY6 (BJ5465) ( 4, A and B). Integration was confirmed by PCR ( 4, C). The established strain will be transformed by pJN58-RAD24 or pJN58-RFC1 plasmid and tested for clamp loader complex expression. Since this method will use only one episome (circular plasmid), much less plasmid-loss, and much higher yield of the five-subunit complex are expected.
Post invasion DNA synthesis by Pol η. DNA strand invasion by Rad51 and extension of the ssDNA end by DNA polymerase η was analyzed. First, labeled synthetic oligonucleotide (16-mer, red arrows above gel in Fig. 5) was incubated with Rad51 and then with template DNA (black lines in Fig. 5) to produce molecules that mimicked the recombination intermediates, the D-loop (DL) and a control substrate (C2). Then, purified 6-His tagged Pol η was added to start DNA synthesis from the labeled oligonucleotides. Products were analyzed by 12% denaturing polyacrylamide gel electrophoresis, containing 8M urea. As shown in 5, Pol η extended the 16-mer primer to the full length (61mer) when C2 was used as the template. When D-loop was used for testing, Pol η was able to initiate the extension efficiently. However, the extension was almost completely blocked at the site where the extension hits the end of D-loop (31-mer product as indicated in 5). This result suggests that yeast Pol η may be less efficient in extending D-loop.
Previous report showed that human Pol η efficiently extended the DL, but these reactions contained approximately a 60-fold excess of enzyme (51), which is much higher than the concentration tested in this experiment (Fig. 5). It was previously reported that the presence of Rad51 inhibit D-loop extension by masking the 3'-OH accessibility to the polymerase (41). However, D-loop extension by Pol η in the presence of Rad51 was observed in our tests. This point will be further investigated for clarification.
Optimization of 9-1-1 clamp expression and purification
Since the expression level varies among the three components of 9-1-1 clamp, it is likely that some unfunctional partial complex may form during reconstitution. Based on this logic, new single plasmid construct which allows us to co-express all the three genes will be tried ( 6, A). To express soluble 9-1-1 complex in E. coli, Rad17, Mec3, and Ddc1 will be overexpressesed in response to IPTG induction at 16ºC. Preliminary experiment showed that this method worked well and 9-1-1 complex could be purified under non-denaturing conditioning (data not shown). In addition, another modification will be introduced to this constructs to express GST-fusion of Ddc1 protein, which will further benefit the purification.
Optimization of Rad24-RFC and RFC expression and purification Preliminary experiments shown above have been done with three plasmid system that co-express five subunits of Rad24-RFC complex (Rad24, Rfc2-Rfc5). Due to the high plasmid lost rate during yeast culture, the three-plasmid system seems to be not efficient to express the five-subunit protein complexes. The more plasmids are transformed into yeast cell, the more difficult to maintain all the plasmids. It has been reported that expression of RFC1, 2, 3, 4, and 5 genes on two plasmids gives higher level of protein yield (REF). To elevate protein yield, four subunit genes (RFC2-5) will be cloned onto pESC-Trp vector ( 6, B) and transformed into yeast TSY6 (BJ5465) together with pJN58-RAD24 plasmid.
Clamp loading assays In vitro experiments that can detect loading of the PCNA and 9-1-1 clamp onto the recombination intermediates will be performed by using the synthetic DNA substrate (Joint Molecule) which mimics a recombination intermediate at the stage of DNA strand invasion (Fig.7.). The structure of the Joint Molecule is similar to that of the D-loop that is used in Pol η polymerase assay, except that the Joint Molecule has a longer invading ssDNA that has a dsDNA region at the non-invading end. Goal of the experiment is to detect loading of the clamp on discrete dsDNA regions in the Joint Molecule (Fig. 7, sites "a" to "d"). Most importantly, I hope to detect the clamp that is loaded inside of the D-loop (site "c"), which is likely to affect the polymerase that mediate post invasion synthesis.
In order to differentiate between these four possible loading sites, dsDNA ends of the synthetic Joint Molecule will be modified with biotin and conjugated with streptavidin ( 7, hutched circles). This modification can block the loaded clamp from sliding off from specific dsDNA region (18, 43, 44). One of the three dsDNA ends in Joint Molecule will be modified at a time (Fig. 7, A, B, and D), so that three Joint Molecules will be prepared (substrates JM-A, JM-B, and JM-D). Another Joint Molecule will also be prepared that receives no modification (JM-0). By using these blocked substrates, the clamp can be selectively trapped at one of the three dsDNA regions. Most importantly, to identify the clamp at site "c", a cutting site of restriction enzyme (Kpn I) was added to the internal dsDNA region of the Joint Molecule (Fig.7.). Cleavage at this site can release the clamp from position "C".
Experimental conditions for in vitro clamp-loading have been established by several groups (18, 19, 45). 32P- labeled Joint Molecules are first incubated with RPA and then with the clamp loader and its cognate clamp in the presence of ATP. Since the sliding clamps have His6-tag, Joint Molecules associated with clamp will be captured by Ni-beads and quantified by its radioactivity (Fig.8.). If the clamp is loaded on the site "A", "B", or "D' of the Joint Molecules (Fig.7.), only the Joint Molecule with streptavidin at A, B, or D site (JM-A, JM-B, and JM-D) should remain associated with clamp, otherwise they will slide off from the substrate. If clamp is loaded on the site "c", all Joint Molecules should be captured with Ni beads. Even the streptavidin-free Joint Molecule (JM-0) should be associated with clamp on site "C" (Fig. 12). Specific loading on site “C” will be confirmed by restriction digestion at site "C", which should specifically release the labeled DNA from clamp (Fig. 8). Therefore, the amount of the Joint Molecule that contains the clamp at site "C" will be quantified as the radioactivity of the JM-0 which is co-precipitated with the clamp and released by cutting at C (Fig. 8). By using these methods, we will determine the relative loading efficiencies of PCNA and the 9-1-1 clamp in distinct sites ("a" to "d") of the Joint Molecule. Based on previous reports, the prefered site for the 9-1-1 clamp loading is dsDNA with a 5'-recessed end (18, 20), which corresponds the site "a" in Fig 7. PCNA is preferentially loaded at the 3'-primer template junction, which does not exist in the Joint Molecule. However, past studies that focused on DNA replication and the damage checkpoint did not specifically analyze for recombination intermediates. Therefore, the clamp-loading specificity on the Joint Molecule is not known. This experiment will be first analysis to evaluate how recombination intermediate is recognized and complexed with the PCNA and 9-1-1 clamps. Clamp loading on site "b" or "c", will be a novel discovery. Especially when a clamp is loaded on site "c", such a clamp is likely to affect the polymerase that mediate post-invasion DNA synthesis.
Effects of loaded clamp on polymerase activity If clamp loading is detected at the site "c", its effects on Pol η and Pol ζ activity will be examined. To do this, either PCNA or 9-1-1 clamp
will be loaded on the Joint Molecule that has no streptavidin block (JM-0), producing the Joint Molecule that has a clamp on the site "c" (Fig. 8). Then, the clamp-JM-0 complex will be pulled down with Ni-beads as described above and mixed with Pol η or Pol ζ to start DNA post-invasion DNA synthesis. Labeled DNA product, which is produced by DNA synthesis, will be analyzed by denaturing polyacrylamide gel electrophoresis (Fig.8.). As a control, clamp-free Joint Molecule will be mixed with polymerases to see the effect of the clamp which is loaded on the site "c".
Polymerase assay on plasmid based Joint Molecule. The advantage of using short synthetic DNA substrate is that their structure could be easily manipulated by changing sequence of synthetic DNA. Alternatively, more native DNA substrates may be used to avoid oligonucleotide-specific artifacts. Such substrate will be produced in a covalently circular plasmid and 32P-abeled synthetic ssDNA (Fig. 9). The ssDNA will be first incubated with Rad51 or Dmcl and then mix with a plasmid to produce D-loop ( 9). Recombinase will mediate DNA strand invasion and promotes the formation of Joint Molecule. The Joint Molecule will be separated from free oligonucleotide substrate by spin-column gel-filtration through Sepharose S-400 (46). Then His-tagged PCNA or His-tagged 9-1-1 clamp will be loaded, and the clamp-Joint Molecule complex will be pulled down by Ni beads as described above. Loading on the specific sites (A, B, or C) of the Joint Molecule could again be differentiated by selectively blocking and by cutting by restriction enzymes at sites A-C (Fig. 9). In case the clamp is loaded at site "c", extension of JM-clamp complex by polymerases will also be analyzed similarly as above.
1. Optimization of 9-1-1 clamp and Rad24-RFC2-5 complex( 9-1-1clamp loader ) expression and purification
2. Cloning, expression and purification of RFC1-5(PCNA clamp loader)
3. Polζ cloning, expression and purification
1. Clamp loading assays using newly purified protein complexes onto C2 and joint molecule substrate
2. Test of Polζ activity with synthetic DNA substrate mimicking HR intermediate.
1. DNA polymerase activity of Pol η and Pol ζ on synthetic DNA substrate with PCNA or 9-1-1 clamp loaded on.
2. Polymerase assay with plasmid based testing system.
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