Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress

Abstract

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Figure 1.

Replication characteristics of minimally licensed DNA. (A) Sperm nuclei were incubated in X. laevis egg extract supplemented with biotin-16-dUTP. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. After 30 min, DNA was isolated and spread on glass slides, and the biotin-labeled tracks were detected with fluorescent antibodies. The distance between the center points of adjacent tracks of labeled DNA was measured. (B) Sperm nuclei were incubated in X. laevis egg extract containing α-[³²P]dATP, plus or minus aphidicolin and/or caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. At the indicated times, the total amount of DNA synthesized was measured.

Figure 2.

Caffeine allows the initiation of extra forks on maximally licensed DNA. (A) Sperm nuclei were incubated in X. laevis egg extract, plus or minus aphidicolin and/or caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. At 40 min, chromatin was isolated and immunoblotted for Cdc45. Coomassie-stained histones are shown as a loading control. (B) Sperm nuclei (400 ng DNA) were incubated in different volumes of egg extract supplemented with 40 μM aphidicolin plus caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. At 40 min, chromatin was isolated and immunoblotted for Cdc45. (C) Sperm nuclei were incubated in egg extract supplemented with biotin-16-dUTP and caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. At 30 min, DNA was isolated and spread on glass slides, and the biotin-labeled tracks were detected with fluorescent antibodies. The distance between the center points of adjacent tracks of labeled DNA was measured.

Figure 3.

Maximally licensed DNA can undergo premature nascent strand fusion in the presence of caffeine. (A) Model for the use of additional dormant origins in extract treated with aphidicolin and caffeine. A small segment (∼30 kb) of chromosomal DNA is shown, which normally supports initiation from three origins. Candidate replication origins with bound Mcm2–7 are shown as gray circles. Nascent strands are shown as double-headed arrows. On maximally licensed DNA (left), nascent strands initiate from dormant origins and rapidly fuse. On minimally licensed chromatin (right), there are no dormant origins so the first strand fusion events occur at the most closely spaced origins. (B and C) Sperm nuclei were incubated in X. laevis egg extract, plus or minus aphidicolin and/or caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. i–iii, maximally licensed DNA; iv–vi, minimally licensed DNA. i and iv, no additions; ii and v, plus caffeine and aphidicolin; iii and vi, plus caffeine. At the start of S phase, the extract was supplemented with α-[³²P]dATP, which was chased with unlabeled dATP after either 2 min (i, iii, iv, and vi) or 5 min (ii and v). At different times thereafter DNA was isolated, separated by alkaline agarose gel electrophoresis, and autoradiographed. The times for i were every minute from 27–36 min. The times for ii were 31, 33, every minute from 35–45, 48, 51, and 54 min. The times for iii were every minute from 23–32 min. The times for iv were every minute from 23–30, 32, 34, 36, and 40 min. The times for v were every 2 min from 32–50, 55, and 60 min. The times for vi were every min from 23–30, 32, 34, 36, and 40 min. Molecular weight markers (λ-HindIII) are shown to the left (sizes in kilobases). The autoradiographs are shown in B. The x-ray film was then scanned and the density of label in each lane quantified. The scans for each lane are shown in C (earliest times at the top, slowest migration to the right). The migration of λ-HindIII is shown by ticks at the bottom.

Figure 4.

Roscovitine blocks premature nascent strand fusion. (A) Model for the effect of roscovitine on dormant origin firing. The model is the same as the maximally licensed DNA shown in Fig. 3 A, except that roscovitine is added shortly after the first origins fire (right), thereby preventing the dormant origins from firing. (B) Sperm nuclei were incubated in X. laevis egg extract supplemented with α-[³²P]dATP. At 25 min (left), or 30 min (right), extract was optionally supplemented with aphidicolin, caffeine, and/or roscovitine. At the indicated times, the total DNA synthesis was measured. (C) Sperm nuclei were incubated in X. laevis egg extract supplemented with aphidicolin and caffeine. At 25 min, the extract was supplemented with α-[³²P]dATP minus (i and iii) or plus (ii and iv) roscovitine. At 30 min, extract was supplemented with unlabeled dATP. At the indicated times, DNA was isolated, separated by alkaline agarose gel electrophoresis, and autoradiographed. End-labeled λ-HindIII was run as molecular weight standards. The autoradiographs are shown in i and ii. The x-ray film was then scanned and the density of the label in each lane was quantified. The scans for each lane are shown in iii and iv (earliest times at the top; slowest migration to the right). The migration of λ-HindIII is shown by ticks at the bottom.

Figure 5.

Minimally licensed DNA is sensitive to a range of replication inhibitors. Sperm nuclei were incubated in X. laevis egg extract supplemented with α-[³²P]dATP plus or minus caffeine. To produce minimally licensed DNA, extract was supplemented with geminin shortly after sperm addition. At 5 min, extract was optionally supplemented with 500 μM mitomycin C, 200 μM etoposide, or 4 ng/μl actinomycin D. At the indicated times, the total amount of DNA synthesized was measured.

Figure 6.

Knockdown of Mcm7 in C. elegans causes hypersensitivity to HU. C. elegans were grown on plates plus or minus bacteria expressing anti-Mcm7 siRNA (2%) and ±9.5 mM HU. After 7 d, plates were examined for worm growth. (A) The number of F1 (first) and F2 (second) generation worms in standardized areas was counted, ±SD. (B) Representative picture of a plate, in each case showing one adult F1 worm. Closed arrowhead, F1 adult; open arrowheads, F2 larvae. Note the absence of F2 worms in the sample containing both HU and anti-Mcm7 siRNA. Bar, 0.2 mm.

Figure 7.

Model for the use of dormant origins on chromosomal DNA. (A) A small segment of chromosomal DNA is shown with a single molecule of ORC bound to it. Distributed around the ORC are several Mcm2–7 double hexamers (hexagons), which each represent a candidate origin. The collection of Mcm2–7 around the ORC are designated a single license group. (Bi) Once initiation occurs in a given license group it generates a weak caffeine-sensitive checkpoint signal that inhibits initiation from any other Mcm2–7 within that license group. (Ci) As the fork replicates the DNA, uninitiated Mcm2–7 at dormant origins are displaced from the DNA. (Bii) The rightward fork stalls, potentially generating a strong global checkpoint signal. (Cii) One of the dormant origins within the license group escapes inhibition and initiates, thus, allowing DNA to the right of the stalled fork to be replicated. The escape from inhibition may be stochastic, or may occur as a result of attenuation of the checkpoint signal after DNA repair. (D) Completion of DNA replication.