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Sister-chromatid cohesion is essential for proper chromosome segregation and faithful transmission
of the genome during the cell cycle (Morales and Losada， 2018; Uhlmann， 2016). Failure to estab-
lish or resolve cohesion in a timely manner leads to genomic instability and aneuploidy. Sister-chro-
matid cohesion is mediated by cohesin， a ring-shaped ATPase machine that consists of SMC1A，
SMC3， RAD21， and either STAG1 or STAG2 in human somatic cells (Haarhuis et al.， 2014;
Losada and Hirano， 2005; Nasmyth and Haering， 2009; Onn et al.， 2008; Peters et al.， 2008;
Zheng and Yu， 2015). Cohesin rings topologically entrap DNA to generate physical linkages
beteen sister chromatids and enable cohesion. Cohesin regulates other chromosome-based pro-
cesses， such as DNA repair， transcription， and chromosome folding (Merkenschlager and Odom，
2013; Wu and Yu， 2012). These other functions of cohesin likely also involve the topological entrap-
ment of chromosomes or possibly the extrusion of DNA loops (Barrington et al.， 2017;
Davidson et al.， 2016; Haarhuis et al.， 2017).
Cohesin is loaded onto chromosomes in telophase and G1 by the SCC2/4 plex (NIPBL/MAU2
in humans)(Ciosk et al.， 2000; Gillespie and Hirano， 2004; Takahashi et al.， 2004; Tonkin et al.，
2004; Watrin et al.， 2006). Before DNA replication， the chromosome-bound cohesin is dynamic and
is actively removed from chromosomes by the cohesin-releasing factor WAPL ith the help of thescaffolding protein PDS5A or PDS5B (Chan et al.， 2012; Kueng et al.， 2006; Lopez-Serra et al.，
2013; Ouyang and Yu， 2017; Ouyang et al.， 2013; Ouyang et al.， 2016). During DNA replication
in S phase， a pool of cohesin is converted to the cohesive form， hich stably associates ith chromo-
somes and mediates sister-chromatid cohesion (Gerlich et al.， 2006; Kueng et al.， 2006). In human
cells， cohesion establishment requires the acetylation of SMC3 by the acetyltransferases ESCO1 and
ESCO2 and subsequent recruitment of sororin， hich antagonizes WAPL to stabilize cohesin on
chromosomes (Alomer et al.， 2017; Hou and Zou， 2005; Nishiyama et al.， 2010; Ouyang et al.，
2016; Rankin et al.， 2005; Rolef Ben-Shahar et al.， 2008; Roland et al.， 2009; Unal et al.， 2008;
Zhang et al.， 2008a).
The checkpoint kinase proteins Mec1 and Rad53 are required in
the budding yeast， Saccharomyces cerevisiae， to maintain cell
viability in the presence of drugs causing damage to DNA or
arrest of DNA replication forks1±3. It is thought that they act by
inhibiting cell cycle progression， alloing time for DNA repair to
take place. Mec1 and Rad53 also slo S phase progression in
response to DNA alkylation4， although the mechanism for this
and its relative importance in protecting cells from DNA damage
have not been determined .Here e sho that the DNA-alkylating
agent methyl methanesulphonate (MMS) profoundly reduces the
rate of DNA replication fork progression; hoever， this moderation
does not require Rad53 or Mec1. The accelerated S phase in
checkpoint mutants4， therefore， is primarily a consequence of
inappropriate initiation events5±7.Wild-type cells ultimately plete
DNA replication in the presence of MMS. In contrast，
replication forks in checkpoint mutants collapse irreversibly at
high rates. Moreover， the cytotoxicity of MMS in checkpoint
mutants occurs speci?cally hen cells are alloed to enter S
phase ith DNA damage. Thus， preventing damage-induced
DNA replication fork catastrophe seems to be a primary mechanism
by hich checkpoints preserve viability in the face of DNA
To ensure that a plete set of the eukaryotic genome is
precisely duplicated during the limited period of S phase in
every cell cycle， DNA replication initiates at a number of
replication origins on chromosomes (Gilbert， 2001; Bell and
Dutta， 2002). As each chromosome region replicates in a
specific period ithin S phase， timing of origin activation
must be regulated. Although e have a groing understanding
of protein factors involved in initiation and elongation of
replication， the mechanisms of origin activation at the chromosome
level are yet to be clarified in detail. Thus， it is
important to determine locations of all replication origins on
chromosomes. Hoever， only small numbers of replication
origins have so far been identified in most organisms other
than budding yeast Saccharomyces cerevisiae (MacAlpine and
Bell， 2005).
The process of initiation of replication at individual replication
origins is posed of to major steps， licensing of
replication origins in G1 phase and activation of the origins in
S phase. In G1 phase， pre-replicative plexes (pre-RCs) are
formed at replication origins (Bell and Dutta， 2002; Kearsey
and Cotterill， 2003). This requires binding of the origin
recognition plex (ORC) to a replication origin， folloed
by assembly of the minichromosome maintenance (MCM)
plex， depending on the loading factors， Cdc6/Cdc18 and
Cdt1 (Diffley et al， 1994; Bell and Dutta， 2002). Although pre-
RC formation is essential for initiation of replication， it is not
in itself sufficient. Origin activation in S phase is regulated by
to conserved protein kinases， cyclin-dependent protein
kinase (CDK) and Cdc7–Dbf4 protein kinase (Dbf4-dependent
kinase， DDK). These kinases are required for assembly of
several other protein factors， including Cdc45 and GINS onto
pre-RCs. This may lead to activation of MCM helicase and
origin DNA uninding， and the replication machinery is
established through assembly of RPA and DNA polymerases
onto the single-stranded DNA (Bell and Dutta， 2002).
Although proteins involved in initiation of replication are
conserved among eukaryotes， the nucleotide sequences of
replication origins are very diverse among organisms
(Gilbert， 2001)， mainly because of differences in DNA-binding
properties of ORCs. In budding yeast， ORC recognizes the
specific sequence called the ARS consensus sequence (ACS).
In contrast， no clear consensus sequence has been found in
origins in fission yeast， Schizosaccharomyces pombe (Clyne
and Kelly， 1995; Dubey et al， 1996; Okuno et al， 1999)，
although AT-rich sequences to hich ORC preferentially
binds are required (Chuang and Kelly， 1999). Requirements
for specific sequences bee less clear in multicellular
organisms such as metazoans， and ORC exhibits little sequence
specificity in DNA binding in vitro (Vashee et al， 2003;
Remus et al， 2004). Therefore， it is important to determine the
locations of ORC binding and DNA synthesis experimentally.
Genome-ide analyses of replication kinetics and distribution
of ORC and MCM proteins using DNA microarrays have
Received: 24 July 2006; accepted: 8 January 2007; published online:
15 February 2007
*Corresponding author. Department of Biology， Graduate School of
Science， Osaka University， 1-1， Machikaneyama-cho， Toyonaka， Osaka
560-0043， Japan. Tel.:t81 6 6850 5432; Fax:t81 6 6850 5440;
E-mail: [email protected]
5Present address: The Scripps Research Institute， North Torrey
Pines Road， La Jolla， CA ， USA
6Present address: Department of Biological Chemistry and Molecular
Pharmacology， Harvard Medical School， 240 Longood Avenue，
Boston， MA 02115， USA
The EMBO Journal (2007) 26， 1327–1339 |& 2007 European Molecular Biology Organization | All Rights Reserved 0261-4189/07
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