Visualization of Altered Replication Dynamics after DNA Damage in Human Cells

Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase. Eukaryotic cells respond to DNA damage within the S phase by activating an intra-S checkpoint: a response that includes reducing the rate of DNA synthesis. In yeast cells this can occur via checkpoint-dependent inhibition of origin firing and stabilization of ongoing forks, together with a checkpoint-independent slowing of fork movement. In higher eukaryotes, however, the mechanism by which DNA synthesis is reduced is less clear. We have developed strategies based on DNA fiber labeling that allow the quantitative assessment of rates of replication fork movement, origin firing, and fork stalling throughout the genome by examining large numbers of individually labeled replication forks. We show that exposing S phase cells to ionizing radiation induces a transient block to origin firing but does not affect fork rate or fork stalling. Alkylation damage by methyl methane sulfonate causes a slowing of fork movement and a high rate of fork stalling, in addition to inducing a block to new origin firing. Nucleotide depletion by hydroxyurea also reduces replication fork rate and increases stalling; moreover, in contrast to a recent report, we show that hydroxyurea induces a strong block to new origin firing. The DNA fiber labeling strategy provides a powerful new approach to analyze the dynamics of DNA replication in a perturbed S phase. Many types of DNA damage can cause mutations in the genome of a cell, not only by direct mutagenesis but also by generating lesions that are processed into mutations when DNA is replicated during S phase. Mechanisms that guard against this include multiple DNA repair systems and also cell cycle checkpoints that coordinate cell cycle progression with the DNA damage response (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2628) Google Scholar). One such checkpoint acts within the S phase to reduce the rate of DNA synthesis, presumably minimizing the risk of damage being fixed into potentially dangerous mutations before it can be repaired. The reduction in rates of DNA synthesis in the intra-S checkpoint may be due to any of a combination of parameters: the overall number of active origins, the temporal program of origin firing, the rates of movement of all active forks, and the occurrence of “fork stalling” events. Any or all of these parameters may be affected by DNA damage, either as a direct physical result of DNA lesions or via the action of checkpoint proteins. This issue has been addressed in some detail in the budding yeast Saccharomyces cerevisiae, in which replication from specific origins has been examined after treatment with methyl methane sulfonate (MMS) 1The abbreviations used are: MMS, methyl methane sulfonate; HU, hydroxyurea; IR, ionizing radiation; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; Gy, gray(s). 1The abbreviations used are: MMS, methyl methane sulfonate; HU, hydroxyurea; IR, ionizing radiation; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; Gy, gray(s). and hydroxyurea (HU) using a combination of Southern blot, two-dimensional gel, and density transfer analyses of replication intermediates. These techniques can separate effects on origin firing from effects on fork rate, at least on a population level, and they have shown that origin firing is blocked in response to MMS or HU (2Tercero J.A. Diffley J.F.X. Nature. 2001; 412: 553-557Crossref PubMed Scopus (557) Google Scholar, 3Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Nature. 1998; 395: 618-621Crossref PubMed Scopus (359) Google Scholar) and that rates of fork movement are also reduced after MMS damage (2Tercero J.A. Diffley J.F.X. Nature. 2001; 412: 553-557Crossref PubMed Scopus (557) Google Scholar). The block to origin firing in yeast depends on the checkpoint kinases Mec1 and Rad53, whereas the reduction in fork rate appears to be independent of these kinases. Mec1 and Rad53 (homologues of human ATM/ATR and Chk2, respectively) are also central to several other aspects of the S phase checkpoint: the induction of a transcriptional program of damage response genes (4Allen J.B. Zhou Z. Siede W. Friedberg E.C. Elledge S.J. Genes Dev. 1994; 8: 2401-2415Crossref PubMed Scopus (343) Google Scholar, 5Aboussekhra A. Vialard J.E. Morrison D.E. de la Torre-Ruiz M.A. Cernakova L. Fabre F. Lowndes N.F. EMBO J. 1996; 15: 3912-3922Crossref PubMed Scopus (109) Google Scholar), the prevention of irreversible fork stalling after MMS damage (2Tercero J.A. Diffley J.F.X. Nature. 2001; 412: 553-557Crossref PubMed Scopus (557) Google Scholar, 6Lopes M. Cotta-Ramusino C. Pellicioli A. Liberi G. Plevani P. Muzi-Falconi M. Newlon C.S. Foiani M. Nature. 2001; 412: 557-561Crossref PubMed Scopus (618) Google Scholar, 7Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Crossref PubMed Scopus (675) Google Scholar), and the increase of dNTP levels in the cell after damage (8Chabes A. Georgieva B. Domkin V. Zhao X. Rothstein R. Thelander L. Cell. 2003; 112: 391-401Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). It is not clear, however, whether these additional checkpoint responses actually affect the rate of DNA synthesis. S phase responses to DNA damage have also been examined extensively in human cells. However, by contrast to the techniques described above, the standard assay for an S phase checkpoint response in mammalian cells, the radioresistant DNA synthesis assay, simply measures rates of overall DNA synthesis by pulse labeling a population of cells with tritiated thymidine after DNA damage. Because it only measures bulk synthesis, this assay cannot distinguish effects on origin firing from those on either fork movement or fork stalling. Moreover, it is affected not only by intra-S phase changes to DNA synthesis but also by inhibition of the G1-to-S transition. In addition, to correlate the incorporation of tritiated thymidine with DNA synthesis, it is necessary to assume that the specific activity of the endogenous dNTP pools These pools however, be affected by changes in the rates of de synthesis after damage. in the of a of and origins in mammalian the of the techniques to have been used to specific aspects of the mammalian S phase of DNA on an after cells with ionizing radiation to the that origin firing is blocked the of DNA to origins, is reduced after damage A. PubMed Scopus Google Scholar, Google Scholar). of labeled to ongoing forks, also to be reduced but only after higher of block to origin firing after MMS and damage, with fork movement being affected to a and only after PubMed Scopus Google Scholar, Google Scholar). The response to and to be in cells. It is to however, that of of these be it is not to the large and DNA actually and the radioresistant DNA synthesis this assay be by changes to dNTP levels as as cell cycle effects the S phase. a of labeling of DNA in cell that at least of the reduction in labeling that to in an population due to the prevention of S phase via a as to any intra-S phase in replication dynamics H. J.M. A. PubMed Scopus Google Scholar). the of a block to origin firing that is intra-S phase and has been by a two-dimensional of replication in of the in the mammalian genome and This at least that origins be blocked damage within S whereas fork movement to be at least after J.M. H. PubMed Scopus Google Scholar). dynamics and on checkpoint not by the two-dimensional after other of DNA damage such as by However, an approach has been used to origin firing after or HU that This the labeling of of and in cells, an block to the of replication when the cells are with G. EMBO J. 2003; PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). This as a checkpoint-dependent block to origin however, the does not quantitative on the numbers or of affected origins in the labeled can it other parameters such as fork rate or fork all of the techniques described above, those used to replication in on examining replication in of cells. all such may that can be by examining replication forks. all these of using a a DNA strategy has been developed in which all the parameters DNA synthesis during the S phase can be on the of replication as to cell This measures DNA synthesis the of or it is and the can be to The has been used in a of the and changes to replication which occur after a of and and cells as in by in cells into PIPES, with for and in DNA after when the of cells in the S phase. The in in cells, also in by in PBS, and for at least in at The cells in and for in of and A. using a DNA to the at of the MMS and cells with before in at and cells from the for the HU in and to the at of to and DNA cells with for for or for and for In the in the cells with for before DNA damage, with thymidine for to the and in before DNA as described by and A. J. 1998; PubMed Scopus Google Scholar), with the cells and in at The labeled cells in cells, and of cells with of in on a the at and the DNA fixed in and with for several in PBS, and blocked in The at with the in PBS, and for in in in in and in The for with in before in and in using a S is by IR, MMS, and techniques used to cells in S such as or thymidine with replication and are to DNA damage in this cells by and for at which cells are in the S phase. we used such cells to the effects of on overall S phase the cells with of MMS, the MMS and S phase progression by the that S phase in a from a after MMS to after the S phase progression after to or of that cause and K. M. A. 2003; PubMed Scopus Google not cause a slowing of S but in a slowing of S phase progression HU to the cells in the S phase. a slowing of the S phase HU a slowing of S and to with a DNA by MMS and HU but by DNA fiber labeling A. J. 1998; PubMed Scopus Google Scholar) has been in this such that and J.A. J. J. PubMed Scopus Google Scholar), be used to replication within a S phase. In this from the DNA fiber J.A. J. PubMed Scopus Google Scholar) in which replicated DNA is labeled with tritiated the cells are with and on a the DNA from the cells is in the of This DNA is and to the In these all of the DNA in a with DNA the of any or pulse labeling of the S phase cells with and labeled on the DNA that can be as either ongoing forks, origins, or fork stalling The of any after a labeling is to fork rate, whereas the numbers of can changes in the rates of origin firing or fork stalling after DNA damage. DNA fiber after of the examined in to which of DNA synthesis to the overall slowing of S phase by any in fork rates after DNA damage, the cells to of MMS and after of the MMS, labeled with for before DNA fiber The of at least for that fork rates reduced for at least after MMS The of slowing with the MMS but slowing only that reduction of dNTP pools by treatment with hydroxyurea also replication in a cells with high levels of HU the and progression several not contrast to MMS and HU, not cause at that reduce overall S phase in the of labeled after of to after IR, MMS, or DNA fiber labeling can be used to distinguish origins from ongoing using the in replication to damage labeled with and the cells with damage and the by the any origins labeled with and they can be against the number of that with to damage. that origin firing in response to MMS and that the of inhibition the of MMS at to also origin firing, but the response to MMS, this may show a and in origin firing after to not This of after the of and MMS damage, may the of the origins, which are to within this labeling at the of damage. The response of cells to HU also in this origin assay, depletion has been shown to origin firing via the S phase checkpoint in (2Tercero J.A. Diffley J.F.X. Nature. 2001; 412: 553-557Crossref PubMed Scopus (557) Google Scholar, 3Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Nature. 1998; 395: 618-621Crossref PubMed Scopus (359) Google Scholar, C. Diffley J.F.X. Nature. 1998; 395: PubMed Scopus Google Scholar). In higher the S phase checkpoint response to HU has not been but which replication by DNA does the of S phase in cells 2000; PubMed Scopus Google Scholar). for with as and the with together with The of new origins against the ongoing the that origin firing is such that it to the number of origin firing that occur in cells in less It is that new origins but simply not labeled of to for a the of HU the using only HU, a that to by new origin firing Because of a recent an increase in origin firing after treatment of a cell with HU M. F. A. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), this using human of cells, and a inhibition of new origin firing at after IR, MMS, and of the assay described used to in the rate of firing after DNA damage in active replication with a pulse of to DNA damage and the before MMS or This the number of active replication before DNA damage. from to the cells with of and This any new origins actually firing at or at labeled in from ongoing replication in These new origins against the that an they been in all the cells before any DNA damage. that a pulse of MMS a block to origin firing when with the levels in cells origin firing to only a during at least after the MMS In a transient block to origin firing with after only origin firing to The of origin firing also after from an HU replication with and by a high of HU for from HU, the with and new origins within against the with either or MMS damage, origin firing after a HU but less after This is to be an of of after HU the within the cells to allow the origins that to by within and within not at an after MMS and HU but after slowing of replication after MMS damage, which in result from at least of fork DNA damage may a to a of for by of all replication or a to a simply be a of transient stalling at fork in as it DNA such fork stalling does occur within the of a it be in the of which to the pulse of they are These as an number of the of these a of fork stalling after higher MMS the that fork slowing via stalling events. not cause fork stalling, with the of overall fork slowing after damage In the of HU all are by high levels of In levels of HU, however, replication does at reduced and in this is fork stalling, in as as HU and in a to high levels when S phase cells are to or HU This the of all the parameters that the rate of DNA synthesis in mammalian cells during S phase and the in which these parameters are affected by DNA damage. The fiber labeling developed is an on other that have been used to S phase checkpoint responses it changes in the rate of origin firing from changes in the rates of fork movement and fork stalling. this of these parameters can be examined and using the cells with of does not in the S phase 112: PubMed Scopus Google Scholar) or the S phase checkpoint in yeast L. J. Diffley J.F.X. 2003; PubMed Scopus Google Scholar), the only changes in DNA synthesis that are by IR, MMS, or labeling also the of replication dynamics on the of as an of an cell It does not allow any of in to higher or but it does allow potentially effects on a of to be and of IR, MMS, and HU on with that of DNA damage affect replication in levels of ionizing by MMS, or depletion by HU can all the overall progression of S phase. In the of IR, this slowing appears to be due to a but transient block to origin firing. Alkylation by MMS a block to origin firing, but this for after the of the does the block to origin firing after MMS also causes additional changes to a slowing of fork movement and the stalling of for that are not after levels of that block origin firing to a depletion by HU reduces fork movement be HU and the cell of and this is by levels of fork stalling. HU treatment also origin firing, but the block by MMS, this is is of within after a HU the of after of the described in cells, which However, the intra-S phase checkpoint is to be and all in S phase cells, any checkpoint not be cells have been shown to DNA synthesis in response to and MMS damage by reduced the of a S phase checkpoint in these cells PubMed Scopus Google Scholar, M. M. J. J. Nature. 2003; PubMed Scopus Google Scholar). The S phase responses to DNA damage in cells are to the responses in of yeast cells to during S phase in an of S of reduced origin firing R. 2003; PubMed Scopus Google Scholar). 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Nature. 2001; 412: 553-557Crossref PubMed Scopus (557) Google Scholar), it is that fork slowing is checkpoint-independent in mammalian cells, being a direct result of replication DNA levels of MMS damage, these lesions may be by such as repair that they are not by the fiber labeling assay, that they a checkpoint to origin firing. higher levels of MMS, however, repair may and lesions repair may on the This be with the in fork rates at after MMS the lesions presumably be radiation from MMS in that after of to are K. M. A. 2003; PubMed Scopus Google Scholar), these be to be as and DNA are in any when the are However, is also to cause and other DNA In contrast to the lesions by MMS, it that any lesions by are either also to be that they not fork movement for any of or that they are It be to whether this is the and whether fork slowing after MMS damage is also in cells specific checkpoint checkpoint have in specific of damage as as simply slowing the cell origin firing, the from recent the response to HU in cells M. F. A. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In these cells, origin firing not simply by the firing of a origin less but this by the of origins and an increase in the overall density of origin firing. in of the cell types cells and origin firing throughout the genome by HU the used by M. F. A. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) with This may be by the that the cells been for to an of and this may have for cells with a checkpoint response to depletion to reduced to with fork slowing by the cells in response to HU also in cells or cells, HU is to cells in S phase with or fork The cells, however, to show S phase progression and in replication This that these cells may have to for they may a of that has some to It is also that the origins examined by M. F. A. M. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) are from the of replication It is that are blocked after and MMS damage, the response to appears to be and Gy, whereas the response to MMS increases This may be due to the that damage is via and MMS damage via Genes Dev. 2001; 15: PubMed Scopus Google Scholar). It has been that is via a and and of after the a in by Nature. 2003; PubMed Scopus Google Scholar). The mechanism of is but the lesions by not any changes to they by a replication fork or damage repair before a checkpoint response not be In budding yeast and checkpoint by MMS active replication R. J. 2002; PubMed Scopus Google Scholar, J.A. Diffley J.F.X. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. Genes Dev. 2002; PubMed Scopus Google Scholar). in yeast that the of Rad53 in response to HU and MMS some number of K. P. Genes Dev. 2002; PubMed Scopus Google Scholar). However, this is the for of Rad53 on the number of replication J.A. Diffley J.F.X. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of types of DNA damage to and may also the rates of from IR, MMS, or firing appears to after a HU but less after of It also after after are at least for these from a HU may be the transient depletion of causes DNA damage, and the for checkpoint be as as and to in HU, it may to dNTP levels to may to or be processed via It has been that is for the slowing of fork progression that is by or that also cause potentially lesions on DNA J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). by HU are into the they may not after HU and may be as DNA damage, in a checkpoint in the of damage repair the of the MMS may cause the number of generating an ongoing checkpoint for the lesions by However, it is not that lesions are any less MMS in recent of the of that at least a can for after K. M. 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Science. 2002; 297: 599-602Crossref PubMed Scopus (675) Google Scholar), all of the of DNA at the of the S phase. and result in any cells that from such a of all the replication that are by DNA damage and of the that these may to the of of DNA lesions or activating specific aspects of the checkpoint In this include the of replication responses to and such as of action on a V. M. J. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In the the from these may also to the of on the of checkpoint in