Stokes MP , Van Hatten R , Lindsay HD , Michael WM .

T32GM07598 NIGMS NIH HHS , T32 GM007598 NIGMS NIH HHS

	Figure 1. Alkylation damage of DNA activates the DNA damage checkpoint in Xenopus egg extracts. (A) Cycling extracts were prepared and supplemented with sperm chromatin (control), MMS-treated sperm chromatin (MMS), or sperm chromatin and linearized plasmid DNA at 25 ng/μl (DSB). Additionally, where indicated, 5 mM caffeine was also included. Extracts were incubated at RT and examined for entrance into mitosis by DAPI staining of nuclei to visualize nuclear envelope breakdown. An extract was scored as mitotic when >50% of the nuclei had undergone nuclear envelope breakdown. The data are plotted as mitotic delay, which is the difference in time between entrance into mitosis for the control extract and entrance into mitosis for the experimental extracts. Control extracts typically required 55–70 min to enter mitosis. All samples contained 2,000 sperm nuclei/μl. (B) 35S-labeled Chk1 ΔKD protein (Michael et al., 2000) was added to cycling egg extracts along with the following: sperm chromatin and no further additions (-), or sperm chromatin and aphidicolin (100 μg/ml; aphid.), MMS-treated chromatin (MMS), or sperm chromatin and plasmid DNA that had been linearized by restriction enzyme digestion (25 ng/μl; DSB). After a 60-min incubation, samples were recovered and analyzed by SDS-PAGE for mobility shifts of the labeled proteins. The input lane shows the Chk1 ΔKD protein before incubation with Xenopus egg extract.
	Figure 2. MMS-induced trans-inhibition of the replication of undamaged DNA. (A) Replication of control and MMS-treated chromatin in NPE. The HSS of egg extract was supplemented with sperm chromatin (control) or MMS-treated sperm chromatin (MMS). After a 30-min RT incubation to assemble chromatin and form preRCs, 2 vol NPE were added along with [32P]dATP, and samples were recovered after continued incubation for 30, 60, or 90 min. All samples contained 2,000 sperm nuclei/μl after addition of NPE. The replication products were analyzed by agarose gel electrophoresis and exposure of the dried gel to a Phosphorimager screen. The graph shows the quantification of the radioactivity present in a given sample. These values are expressed as arbitrary units, where the value for the control sample at 90 min was set to 100, with all other samples adjusted accordingly. The data shown are from a single experiment, and are representative of four independent experiments. (B) Experimental strategy. When control (undamaged) or MMS-treated chromatin are incubated separately in NPE containing fluorescent nucleotides, the control sample will appear brighter due to its enhanced ability to undergo DNA replication. To ask if the MMS-treated sample inhibits DNA replication in trans, control and MMS-treated chromatin are incubated together in the same NPE. If the inhibition works in cis only, then two populations of fluorescent chromatin will result: a bright population (corresponding to the control template) and a dim population (corresponding to the MMS-treated sample). However, if inhibition can also work in trans, then all chromatin templates in the coincubation are expected to exhibit reduced fluorescence, relative to the control sample alone. (C) Either control, undamaged sperm chromatin (control), or MMS-treated sperm chromatin (MMS), or a 50:50 mixture of both (control + MMS) were incubated for 30 min in HSS. After the 30-min incubation, NPE containing bio-dUTP was added to the reactions. Incubation was performed for an additional 30 min before processing of the samples for detection of bio-dUTP incorporation with fluorescent streptavidin. In the panel labeled control + MMS/gem., MMS-treated chromatin was incubated in HSS with recombinant geminin for 30 min, then combined with control, undamaged chromatin that had been incubated separately in HSS-lacking geminin, also for 30 min. NPE-containing bio-dUTP was then added to the combined sample. Panels labeled Bio-dUTP display signal obtained from staining of the samples with Texas red–conjugated streptavidin, to detect the bio-dUTP, and panels labeled DAPI correspond to DAPI staining of the samples to visualize the DNA. (D) Quantification of the data presented in C. The fluorescent intensity of a minimum of 50 nuclei for each sample from each of two independent experiments was determined using the Scion Image software package from images obtained from a fluorescence microscope (BX51; Olympus) attached to a Spot camera (Diagnostic Instruments, Inc.). The control + MMS/gem. sample is represented by two bars (labeled dim and bright) to provide individual quantification for the two classes of nuclei present in this particular sample.
	Figure 3. The initiation of DNA replication is required to generate the MMS damage signal. (A) Cycling extracts were prepared and supplemented with either PBS (buffer) or recombinant geminin (gem.). The samples were then further supplemented with either sperm chromatin (control DNA), MMS-treated sperm chromatin (MMS DNA) or sperm chromatin and plasmid DNA that had been linearized by restriction enzyme digestion (25 ng/μl; DSB DNA). Entrance into mitosis was determined as in Fig. 1 A, and the data are plotted as in Fig. 1 A. (B) Cycling egg extracts were prepared and supplemented with cyclohexamide and either PBS (buffer), recombinant geminin (250 nM; gem.), or recombinant p27Kip (500 nM). Extracts were further supplemented with MMS-treated chromatin and 35S-labeled Chk1 ΔKD protein. After a 60-min incubation, samples were recovered for SDS-PAGE. The input lane shows the Chk1 ΔKD protein before incubation with Xenopus egg extract. (C) Cycling extracts were prepared and supplemented with cyclohexamide and either control, undamaged sperm chromatin, or MMS-treated sperm chromatin. Chromatin was isolated from these extracts at the indicated times (in minutes), and probed for the presence of Rad17 using anti–Xenopus Rad17 antibodies. Where indicated, the samples also included caffeine (caf., at 5 mM) or recombinant geminin (gem., at 250 nM). The sample labeled no DNA refers to a sample that was processed in the absence of any sperm chromatin addition, showing that Rad17 recovery is dependent on the addition of chromatin. Sperm chromatin was added to 2,000/μl in all samples.
	Figure 4. Replication structures specific for dsDNA replication are required to generate the MMS damage signal. (A) M13 ssDNA was annealed to an oligonucleotide primer, or not, and then incubated in NPE containing [32P]dATP (final concentration of M13 ssDNA was 5 ng/μl). Additionally, M13 ssDNA was alkylated, annealed to a primer, and incubated in NPE at 5 μg/ml. After either 30 or 60 min, aliquots were removed from the reaction and processed for DNA replication analysis on agarose gels. The top portion of the figure shows the gel, and beneath it is a bar graph showing the values obtained after analysis of the scanned gel on a Phosphorimager. After quantification of the data by Phosphorimager analysis, the value for the 60-min time point for M13 ssDNA plus primer was set to 100, and all other data were normalized accordingly. (B) NPE was supplemented with recombinant, bacterially expressed Chk1ΔKD at 5 ng/μl. The reactions were further supplemented with either alkylated M13 ssDNA (ssDNA), alkylated M13 ssDNA containing a preannealed oligonucleotide primer (primed ssDNA), or plasmid DNA that had been incubated previously in 0.5 vol of HSS either in the absence (dsDNA) or presence (dsDNA + gem.) of recombinant geminin (at 250 nM). After 60 min of incubation, samples were withdrawn and analyzed by SDS-PAGE, and immunoblotting for the phosphorylation status of Chk1ΔKD was performed. Chk1ΔKD was detected using a T7 mAb that recognizes the epitope tag supplied by the expression vector used to produce the recombinant Chk1ΔKD. Plasmid DNA was present at 25 ng/μl final concentration, whereas M13 DNA was present at 75 ng/μl final concentration. The input lane shows the Chkl1 ΔKD protein before incubation with Xenopus egg extract.

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