(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Table 1. Strains used in this study

Strain Name	Genotype
AIAy20	ade2–1 ura3–1 his3–11,15 trp1−1 leu2–3,112 can1–100 lys2::HisG Bar1::HisG orc1::HisG pSPB162 MATa
RKySO	ade2–1 ura3–1 his3–11,15 can1–100 Bar1::HisG orc1.:HisG leu2::ORC1 trp1::p404-GAL1–10-ORC3,4 lys2::plys2-GAL1–10-ORC2,5 MATa
RKy61	RKy50 his3::p403-GAL1–10-ORC1c-HA, 6
RKy62	RKy50 his3::p403-GAL1–10-Orc1(K485T)c-HA,6
RKy63	RKy50 his3::p403-GAL1–10-Orc1-dlc-HA,6
RKy64	RKy50 his3::p403-GAL1–10-Orc1-d2c-HA,6
RKy83	RKy63 ura3::pSF321-CDC6
RKy84	RKy64 ura3::pSF321-CDC6
RKy85	RKy63 ura3::pSF323
RKy86	RKy64 ura3::pSF323
RKy87	RKy63 ura3::pSF321-cdc6K114E
RKy88	RKy64 ura3::pSF321-cdc6K114E
RKy90	AIAy20 his3::p403-ORC1
RKy91	AIAy20 his3::p403-orc1-K485T
RKy92	AIAy20 his3::p403-orc1-d1(D569Y)
RKy93	AIAy20 his3::p403-orc1-d2(D569F)
RKy94	ade2–1 ura3–1 trp1–1 leu2–3,112 can1–100 lys2::HisG Bar1::HisG orc1::HisG his3::p403-ORC1 MATa
RKy95	ade2–1 ura3–1trp1–1 leu2–3,112 can1–100 lys2::HisG Bar1::HisG orc1::HisG his3::p403-orc1-d1 MATa
RKy96	ade2–1 ura3–1trp1–1 leu2–3,112 can1–100 lys2::HisG Bar1::HisG orc1::HisG his3::p403-orc1-d2 MATa

into pMDW13 (ORC1,6), pMDW8 (ORC3,4), and pSPB25 (ORC2,5) baculovirus transfer vectors (21). Fragments containing the GAL1–10 promoter-driven ORC genes were then subcloned into the multicloning sites of yeast integrating vectors p404 (to generate p404 Gal1–10 ORC3,4), p405 (to generate p405 Gal1–10 ORC2,5), and p403 (to generate p403 Gal1–10 ORC1,6). The lys2 gene from pRS317 cut with PvuII was subcloned into p405 Gal1–10 ORC2,5 cut with Tth111 I and XhoI to generate pLys2 Gal1–10 ORC2,5. A triple hemagglutinin (HA) tag was introduced at the C terminus of ORC1 in the overexpressing construct to yield p403 Gal1–10 ORC1c-HA,6. To test complementation, mutants were subcloned into p403-ORC1 (with the endogenous ORC1 promoter) and integrated into AIAy20. The plasmid-borne copy of wild-type ORC1 was selected against by plating on media containing 5-fluoroorotic acid (5-FOA). Mutants were also subcloned into pMDW13 for protein expression. CDC6 plasmids pSF320-CDC6 and pSF320-Cdc6K114E contain the CDC6 gene with an N-terminal 10XHis tag and a C-terminal 3XHA tag under the control of the Gal1–10 promoter. The control vector pSF322 expresses only the 3XHA tag.

Screen for Lethal When Overexpressed Mutants. Four oligonucleotides were used that contain degenerate sequences at the three positions of each of the four codons of the Walker B “DELD” sequence (amino acids 566–569) of Orc1p. The sequences of these oligonucleotides (which hybridize to the sense strand) are as follows: ORC1B1, 5′TTCGTTACCA TGGCATCGAG TTCFENCAAC AAGACTACAA TGGTTTTC-3′; ORC1B2, 5′-TTCGTTACCA TGGCATCGAG FENGTCCAAC AAGACTACAA TGGTTTTC-3′; ORC1B3, 5′-TTCGTTACCA TGGCATCFEN TTCGTCCAAC AAGACTACAA TGGTTTTC-3′; ORC1B4, 5′-TTCGTTACCA TGGCFENGAG TTCGTCCAAC AAGACTACAA TGGTTTTC-3′; where F= G(40%), C(40%), A(20%), and T(0%); where E=G(20%), C(20%), A(30%), and T(30%); and where N=G(25%), C(25%), A(25%), and T(25%). These ratios were chosen to minimize amino acid bias and stop codons, similar to ref. 22. These oligonucleotides were used to PCR amplify the ATP binding domain of ORC1. The mutant PCR products were first cloned into p403-ORC1c-HA (containing a triple HA tag at the C terminus of ORC1). The pool of mutant orc1 genes was then ligated into p403 Gal1–10 ORC1c-HA,6. Plasmids were prepared from individual transformants from the ligation, and were individually tested by integration into RKy50 and streaking on plates containing 2% Galactose.

Protein Purification. ORC mutant complexes were expressed by using baculovirus-infected cells and purified as described (14).

Chromatin Immunoprecipitation (CHIP). ChIP was performed as described (1) with minor modification. For ORC ChIP, a rabbit polyclonal ORC antibody was used. For MCM ChIP, a monoclonal antibody that recognizes all six MCM subunits was used. Incubation time for this antibody was 6 h, after which protein G beads were added and incubated for an additional hour. PCR was performed for 28 cycles on 1/50 of the immunoprecipitates, and on 1/500 of the input material. Quantification was performed by using the Molecular Dynamics Fluorimager and IMAGEQUANT software. To assay loading of MCM proteins during orc1 mutant overexpression, cells were grown in 2% raffinose and arrested with 10 µg/ml nocodazole. After 3 h in nocodazole, galactose was added to 2% to induce ORC overexpression or glucose was added to 2% to repress expression. After an additional 90 min, cells were washed three times and resuspended in media containing 50 ng/ml alpha factor and either 2% galactose or 2% glucose. Cells were fixed for ChIP after >95% of cells were in G₁.

ATP Hydrolysis Assays and DNase I Protection Assays. ORC ATP hydrolysis was monitored by using TLC as previously described (14). Hydrolysis reactions contained 1 µg ORC, 50 mM Hepes (pH 7.6), 150 mM KCl, 5 mM MgOAc, 1 mM EDTA, 1 mM EGTA, 0.02% Nonidet P-40, and ATP as indicated. All reactions included 0.5 µCi alpha [³²P] ATP. Total reaction volume was 13.3 µl. Aliquots of 1.5 µl were removed and added to 0.38 µl 2% SDS over a time course of 3 h.

DNase I protection assays were performed as described (21). Each reaction contained 50 ng ORC, 50 ng poly(dGdC) competitor DNA, and ≈5 fmol of DNA probe derived from pARS1/WT cut with EcoRI and HindIII (radiolabeled on the T-rich strand of the ARS concensus sequence).

Results

Dominant Lethal Alleles Within ORC1. To address the role of Orc1p ATP hydrolysis, we sought mutants in the Walker B motif of

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