Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells - PubMed (original) (raw)
Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells
Nasrollah Saleh-Gohari et al. Nucleic Acids Res. 2004.
Abstract
DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.
Figures
Figure 1
The SCneo substrate for conservative HR in SW480SN.3 cells. (A) Schematic illustration of the SCneo recombination substrate that contains two non-functional copies of the neoR gene in the SW480SN.3 cell line. (B) Following an I-SceI-induced DSB, a functional neoR gene can be gained by HR through intrachromatid pairing or sister chromatid pairing. Intrachromatid pairing is possible throughout the cell cycle and sister chromatid pairing is only possible during late S or G2/M phases of the cell cycle. A short tract gene conversion HR event gives a 6.7 kb product, while SCE or long tract gene conversion restores a neoR gene within a 10 kb product (14). Non-conservative SSA does not produce a functional neoR gene. (C) Southern blot of DNA isolated from the SW480SN.3 cell line containing one copy of the intact SCneo substrate, using S2neo as probe.
Figure 2
Conservative HR repairs DSBs primarily in cells in the S phase of the cell cycle. (A) Cell cycle profiles of SW480SN.3 cells arrested in the S, M or G1/G0 phase of the cell cycle with thymdine (2 mM), nocodazole (40 ng/ml) or serum starvation (1%), respectively. Cells arrested in the cell cycle were transfected with either pCMV3xnlsI-SceI or pEGFP-C2 vector for 5 h and at the same time kept in the given phase of the cell cycle. Cells were maintained in the arrested stage for an additional 7 h before continuation of recombination assay or GFP expression assay. (B) HR induced with or without a DSB at different phases of the cell cycle. (C) GFP expression from the CMV promoter at different phases of the cell cycle.
Figure 3
Similar spectra of recombinants in HR repair of DSBs at different phases of the cell cycle. Spectrum of DSB-induced recombinant clones that arose in the S, M or G1/G0 phase of the cell cycle. Numbers indicate the number of clones within each group.
Similar articles
- Dependence of DNA double strand break repair pathways on cell cycle phase in human lymphoblastoid cells.
Takashima Y, Sakuraba M, Koizumi T, Sakamoto H, Hayashi M, Honma M. Takashima Y, et al. Environ Mol Mutagen. 2009 Dec;50(9):815-22. doi: 10.1002/em.20481. Environ Mol Mutagen. 2009. PMID: 19402155 - Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate.
Nikolova T, Ensminger M, Löbrich M, Kaina B. Nikolova T, et al. DNA Repair (Amst). 2010 Oct 5;9(10):1050-63. doi: 10.1016/j.dnarep.2010.07.005. Epub 2010 Aug 13. DNA Repair (Amst). 2010. PMID: 20708982 - DNA repair and chromosomal alterations.
Natarajan AT, Palitti F. Natarajan AT, et al. Mutat Res. 2008 Nov 17;657(1):3-7. doi: 10.1016/j.mrgentox.2008.08.017. Epub 2008 Aug 29. Mutat Res. 2008. PMID: 18801460 Review.
Cited by
- Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells.
Graham JP, Castro JG, Werba LC, Fardone LC, Francis KP, Ramamurthi A, Layden M, McCarthy HO, Gonzalez-Fernandez T. Graham JP, et al. bioRxiv [Preprint]. 2024 Sep 23:2024.09.23.614499. doi: 10.1101/2024.09.23.614499. bioRxiv. 2024. PMID: 39386541 Free PMC article. Preprint. - Origins of cancer: ain't it just mature cells misbehaving?
Cho CJ, Brown JW, Mills JC. Cho CJ, et al. EMBO J. 2024 Jul;43(13):2530-2551. doi: 10.1038/s44318-024-00099-0. Epub 2024 May 21. EMBO J. 2024. PMID: 38773319 Free PMC article. Review. - Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair.
Li G, Yang X, Luo X, Wu Z, Yang H. Li G, et al. Cell Biosci. 2023 Nov 25;13(1):215. doi: 10.1186/s13578-023-01159-4. Cell Biosci. 2023. PMID: 38007480 Free PMC article. - Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant.
Mishra AP, Hartford S, Chittela RK, Sahu S, Kharat SS, Alvaro-Aranda L, Contreras-Perez A, Sullivan T, Martin BK, Albaugh M, Southon E, Burkett S, Karim B, Carreira A, Tessarollo L, Sharan SK. Mishra AP, et al. Cell Death Dis. 2023 Nov 18;14(11):753. doi: 10.1038/s41419-023-06289-8. Cell Death Dis. 2023. PMID: 37980415 Free PMC article. - Targeting DNA-PK.
Novotny JP, Mariño-Enríquez A, Fletcher JA. Novotny JP, et al. Cancer Treat Res. 2023;186:299-312. doi: 10.1007/978-3-031-30065-3_16. Cancer Treat Res. 2023. PMID: 37978142
References
- van Gent D.C., Hoeijmakers,J.H. and Kanaar,R. (2001) Chromosomal stability and the DNA double-stranded break connection. Nature Rev. Genet., 2, 196–206. - PubMed
Publication types
MeSH terms
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials