Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Dec 15;17(24):3017-22.
doi: 10.1101/gad.279003. Epub 2003 Dec 17.

Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein

Mikhail Lomonosov  1 , Shubha Anand, Mahesh Sangrithi, Rachel Davies, Ashok R Venkitaraman
Affiliations

Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein

Mikhail Lomonosov et al. Genes Dev. .

Abstract

How dividing mammalian cells overcome blocks to DNA replication by DNA damage, depleted nucleotide pools, or template-bound proteins is unclear. Here, we show that the response to blocked replication requires BRCA2, a suppressor of human breast cancer. By using two-dimensional gel electrophoresis, we demonstrate that Y-shaped DNA junctions at stalled replication forks disappear during genome-wide replication arrest in BRCA2-deficient cells, accompanied by double-strand DNA breakage. But activation of the replication checkpoint kinase Chk2 is unaffected, defining an unexpected function for BRCA2 in stabilizing DNA structures at stalled forks. We propose that in BRCA2 deficiency and related chromosomal instability diseases, the breakdown of replication forks, which arrest or pause during normal cell growth, triggers spontaneous DNA breakage, leading to mutability and cancer predisposition.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Analysis of rDNA replication by two-dimensional (2D) gel electrophoresis. (A) Genomic structure of the murine rDNA repeat, scaled in bp. The origin of bidirectional replication (OBR) and the replication fork barrier (RFB) flank the 5446-bp ApaL1 restriction fragment analyzed in D (thick line ending with an arrow in the top panel). A filled rectangle marks the hybridization probe used in Figs. 1D and 3A. (B) Replication intermediates detected by 2D gel electrophoresis. Y-shaped DNA junctions at replication forks rise off the diagonal to form the Y-arc. A cone-shaped signal extending from the crest of the Y-arc represents four-way DNA junctions. Positions of unreplicated (UnR) and replicated (R) DNA are marked. (C) First-passage cells were synchronized as described, and released into S phase for 6 h (-HU sample, ie., no HU treatment). HU was then added to 5 mM concentration and samples taken at 3 h (+HU 3h) and 6 h (+HU 6h) during exposure. Nuclei were stained for replication foci (BrdU, 30′ pulse, green), and for DNA (DAPI, blue). Apoptotic changes marked by Annexin V staining do not occur during HU treatment (data not shown). (D) Replication intermediates from replication of the rDNA genes in S phase-synchronized cells, before and during HU treatment for up to 6 h. Slight distortion of the Y-arc in the first panel (+/+, -HU) is due to a tear in the gel. (E) Relative intensities of the Y-arc signal in the different 2D gels (expressed as a densitometric ratio over signal on the gel diagonal to normalize for DNA loading) were plotted as a percentage of the value in S-phase cells before HU treatment. Standard deviations from the mean are too small to be visible.
Figure 2.
Figure 2.
Nascent DNA synthesis and turnover after replication blockage. (A) The experimental protocol used to detect nascent DNA turnover is outlined in the top panel, and explained in the text. The ratio of 3H labeling of nascent DNA over uniform 14C labeling of genomic DNA in synchronized cells before and during HU treatment is plotted as a percentage of the initial ratio. (B) Similarly, the protocol used to determine nascent DNA synthesis is outlined above the results. The ratio of 3H incorporation into nascent DNA over uniform 14C labeling is shown at 0-2 h, 2-4 h, and 4-6 h during the 6-h course of HU treatment as a percentage of the ratio in untreated cells (-HU).
Figure 3.
Figure 3.
Breakdown of rDNA and genomic DNA after replication blockage. (A) PFGE showing broken rDNA in BRCA2Tr/Tr cells before and during HU treatment. The 325-bp probe used to detect fragmentation is the same as that used in Fig. 1D to detect replication intermediates. Molecular-weight markers are shown in Mb. (B) Relative intensity of broken rDNA entering the gel measured by densitometry is plotted in arbitrary units. Standard deviations from the mean value are marked when large enough to be visible. (A,D) Relative amounts of broken chromosomal DNA detected by ethidium bromide staining were measured by densitometry and plotted in arbitrary units. (E) DNA breakage occurs in the ADA or HPRT genomic DNA loci of BRCA2Tr/Tr cells treated with HU under the same conditions used in panel A. (F) DNA breakage detected by ethidium bromide staining is dependent on HU dose. Doses ≥5 mM produce the same effect. Experiments are typical of at least two independent repeats.
Figure 4.
Figure 4.
Checkpoint response to replication blockage. (A) Nuclei stained for phosphorylated histone H3 (P-HisH3, green), a marker for mitotic chromosomes, and DNA (DAPI, blue) before and during HU treatment. Staining after metaphase arrest induced by treatment with nocodazole (+Noc) for 16 h serves as a positive control. (B) Chk2 activation in S phase-synchronized cells before (-HU), during (+HU 240′) and at 2 h and 4 h after removal of HU treatment is shown. Lines mark the migration of unmodified Chk2 and the retarded phospho-Chk2 (P-Chk2) bands. (C) Pulsed-field gel electrophoresis showing broken chromosomal DNA during (+HU) HU treatment, and at various times after removal of HU. (D) Cell cycle recovery after removal of HU. After exposure to 5 mM HU for 4.5 h, cells were released from HU, and cell cycle profiles were determined by flow cytometry. The percentage of cells in the G1, S, and G2/M phases is shown at 5 to 33 h after removal of HU.
See this image and copyright information in PMC

References

    1. Alcasabas A.A., Osborn, A.J., Bachant, J., Hu, F., Werler, P.J., Bousset, K., Furuya, K., Diffley, J.F., Carr, A.M., and Elledge, S.J. 2001. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell. Biol. 3: 958-965. - PubMed
    1. Boddy M.N., Furnari, B., Mondesert, O., and Russell, P. 1998. Replication checkpoint enforced by kinases Cds1 and Chk1. Science 280: 909-912. - PubMed
    1. Carroll S.M., DeRose, M.L., Kolman, J.L., Nonet, G.H., Kelly, R.E., and Wahl, G.M. 1993. Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci. Mol. Cell. Biol. 13: 2971-2981. - PMC - PubMed
    1. Chaturvedi P., Eng, W.K., Zhu, Y., Mattern, M.R., Mishra, R., Hurle, M.R., Zhang, X., Annan, R.S., Lu, Q., Faucette, L.F., et al. 1999. Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Oncogene 18: 4047-4054. - PubMed
    1. Chen C. and Kolodner, R.D. 1999. Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants. Nat. Genet. 23: 81-85. - PubMed

Publication types

MeSH terms