DNA
Replication: Old Game, New Players
Kaveri
Sidhu, Vijay Kumar
Kaveri
Sidhu, Vijay Kumar, Virology Group, International Center for Genetic
Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi- 110067, India
Correspondence
to: Vijay Kumar, J.C. Bose National Fellow,
Virology Group,
International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali
Marg, New Delhi- 110067, India.
Email: vijay@icgeb.res.in
Telephone: +91-11-26742360
Fax:
+91-11-26742316
Received: March 21,
2015
Revised: April 16, 2015
Accepted: April 21, 2015
Published online: June 6, 2015
ABSTRACT
Metazoans utilize
multiple origins of replication in order to replicate their vast genome
faithfully and expeditiously during each cell cycle. However, lesser known are
the salient features defining these origins and their mechanism of selection
for replication process. Here, we provide burgeoning evidences which suggest
that transcription factors indeed play a major role in facilitating licensing
of origins for firing during the S phase.
© 2015 ACT. All
rights reserved.
Key words: DNA replication; Chromatin; Replication origin;
Transcription Factors
Sidhu K, KumarV. DNA
Replication: Old Game, New Players. Journal of
Biochemistry and Molecular Biology Research 2015; 1(2): 32-35 Available from: URL:
http://www.ghrnet.org/index.php/jbmbr/article/view/1127
EDITORIAL
All living things reproduce by faithfully replicating their genetic
material. The process of DNA replication involves unwinding double-stranded DNA
to recruit DNA polymerase complexes at predesignated positions in the genome
called origins of replication (oris) followed by replication of both DNA
strands in opposing directions in a continuous (5’-3’) or discontinuous (3’-5’,
Okazaki fragments) manner finally terminating at a specific site or by merging
of progressing replicating forks. As genome size and complexity rises from
prokaryotes to eukaryotes, the steps regulating replication also increase. For
example, in eukaryotes, initiation of DNA replication is a regulated two-step
process to ensure a single round of replication during a cell cycle (Figure 1).
The first step, origin licensing, involves the sequential assembly of
pre-replicative complex (pre-RC) to the oris in the G1 phase comprising of a
multi-subunit DNA binding origin recognition complex (ORC) that marks ori sites
followed by licensing factors Cdc6, Cdt1 that prevent re-replication and
finally the helicase MCM 2-7. The second step marks the transition to the
pre-initiation complex (pre-IC) in the S phase which involves the loading of
MCM10, Cdc45, GINS, Dbf4, Sld3 and Treslin proteins[1]. Escherichia
coli with a genome size of 4×106 bp replicates its
genome in less than 30 minutes compared to human cells that have only ~8 hours
of S phase to replicate a genome of 3×109 bp, three orders of
magnitude larger[2,3]. Metazoans also face the arduous task of
unpacking their compact and supercoiled DNA to access their genetic information
for replication or transcription. Such enormous discrepancy in their genome
complexity and replication time has been circumvented by utilizing multiple
oris in a tightly regulated and sequentially coordinated manner. Unlike
bacteria and budding yeast, oris in higher eukaryotes are not well-defined
sequences but designated by DNA structural elements and chromatin context which
navigate the interaction between initiator proteins and ori. Oris are generally
G-C rich sequences with origin G rich element (OGRE) that form G-quadruplexes
essential for initiation of DNA replication[4]. There are ~100,000
predicted oris in humans of which ~30,000-50,000 are activated during each cell
cycle[5]. The spare oris perhaps form part of a contingency plan to
support the replication program during extreme growth conditions, stalling of a
progressing replication fork, or specific physiological state such as embryonic
development during which S phase lasts for a short period of time and a lot
more oris are engaged than in somatic cells. Further, an excess of oris may
allow the replication to coordinate with the transcription program and
safeguard against DNA damage[1]. As permissive chromatin environment
is a prerequisite for transcription as well as replication, not surprisingly,
oris are often found in the vicinity of actively transcribed gene promoters[6].
Further, active transcription may influence ori selection in two ways, first,
promoting replication by providing a nucleosome free region to allow assembly
of pre-RC proteins or it may inhibit assembly of replication factors. Gene
density correlates with early replicating segments whereas repetitive elements
associated with heterochromatin, telomeres and fragile sites fall under late
replication in S phase[7].
Early
replication oris are found associated with multiple marks of active chromatin
including DNaseI hypersensitivity, H3K4 trimethylation and even RNA polymerase
II association. Transcription
factors (TFs) may, therefore, form a major class of trans-acting factors that
can exert control on ori licensing, selection and timing of firing as
illustrated briefly in Figure 1. Viruses such as SV40 and bovine papilloma
virus, traditionally used model systems to study eukaryotic replication, are
known to employ viral trans-activator proteins to recruit TFs to their oris to
alter the local chromatin structure[8,9]. Recent spurt of studies in
metazoans strongly support this hypothesis and have established an unequivocal
transcription-independent role for c-Myc in control of DNA replication. Early
expressed c-Myc directly interacts with the components of pre-RC and its
overexpression leads to replicative stress by increasing the density of early
firing oris[10]. Swarnalatha et al[11] deciphered
the molecular underpinnings of c-Myc on the regulation of human β-globin ori.
c-Myc transiently associates with unmethylated E-box on lamin B2 ori and
recruits chromatin modifying enzymes to initiate epigenetic modifications
including hyperacetylation of histones which further enhanced the recruitment
of licensing factors. Forkhead TFs are able to influence replication timing,
independent of their transcription regulation role, by interacting with
initiation factors Cdc45 and ORC to bring about clustering of early oris[12].
Similarly, insertion of an USF binding sequence on either side of the β-globin
replicator in an artificial construct is able to alter the replication timing[13].
In Xenopus eggs, introduction of a TF and its cognate binding site
template was able to form an ori with increased acetylation of histone lysines[14].
Also, the duration of S phase in Drosophila and Xenopus extends
during the late embryonic development due to firing of fewer oris as compared
to firing of almost all oris until the mid-blastula stage[15,16]. In
Drosophila, the activated oris tend to lie in the vicinity of actively
transcribed promoters[17].
Whereas, early
oris are associated with transcriptionally active regions and may be regulated
by early expressing TFs such as c-Myc and Jun/Fos, the temporal positioning of
late oris is considered a result of rate limiting levels of replication
proteins which are cycled from early to late oris[18]. Late ori
associated ORC complexes understandably show correlation with silenced
chromatin marks[19]. In addition to TFs, a host of DNA modifying
enzymes influence replication firing and timing. Histone deacetylase SIR2 acts
a negative regulator of pre-RC formation while HBO1, a histone
acetyltransferase and co-activator of Cdt1, remains essential for replication
licensing[20,21]. In addition to a nucleosome free central initiator
region, histone modifications (H4K20 me1) and nucleosome remodelers (PR-Set7,
SNF2H) also contribute to recruitment of pre-RC components at the ori[22,23].
Interaction of ORC with chromatin and adjacent nucleosomes is a must for ori
firing[5]. Similarly DNA demethylase, TET2, is reported to actively
demethylate DNA to maintain chromatin in active state[24].
Replication proceeds in the nucleus in defined regions called replication foci
which are organized from chromatin loops called replicons consisting of an
average five licensed oris which further associate to form a cluster of
replicons[25]. A single ori is activated from each replicon during a
cell cycle and all oris associated with a cluster of replicons are fired
simultaneously showing a strong correlation between replication timing and
spatial arrangement of oris[5]. Recently, Rif-1 a telomere binding
protein has been identified as a negative regulator of late oris while
promoting early oris through its ability to regulate replication loop
structures[26].
Thus,
replication and transcription can be seen as two different sports with similar
rules being played on the same field with transcription having larger number of
teams while replication having fewer but more adept teams. In order to
economize the cell’s efforts, replication often uses the groundwork laid down
by transcriptional machinery delineated in Table 1. This crosstalk between the
two processes is also mediated by sharing of common players such as TFs,
histone modifying enzymes or nucleosome remodeling complexes. However, a single
conflict in their schedules has dire consequences on cell fate, leading to
torsional stress, fork stalling and DNA double strand breaks which is otherwise
prevented by stringent checkpoints in the cell cycle. The importance of
faithful replication of genetic material is evident from the mostly lethal
phenotype exhibited by mutants of the components of replication machinery.
Nevertheless, some pathologies resulting from defects in DNA replication do
exist such as Meier-Gorlin syndrome caused by mutations in the pre-RC
components ORC1, ORC4, ORC6, Cdt1 and Cdc6 resulting in compromised DNA
replication rate and manifestation of primordial dwarfism symptoms such as
delayed development of kneecaps, ears and microcephaly[27]. Besides,
there is a well-established link between deregulation of replication control
and cancer evident in some oncogenic viruses such as hepatitis B virus and
human papilloma virus. Oncoproteins encoded by these viruses, such as HBx and
E7, induce overexpression of replication factors PCNA, Cdt1, Cdc6 which
correlates with re-replication or activation of incorrect oris leading to
polyploidy, a characteristic of malignancy[28,29]. The burgeoning
evidences point towards regulation of replication by TFs and tempts us to
speculate beyond the existing paradigm relating to DNA replication. The answers
that have eluded us so far include criteria for selection of an ori during
replication, mechanism distinguishing early vs. late oris and regulation of
some excessive licensed oris in the S phase. The domain of late replicating
heterochromatic regions and fragile sites remains largely untouched. Besides,
the existence of distinct ori fingerprints during embryonic development, growth
and differentiation or even viral infections is most likely a resultant of the
coordination between ori firing and gene expression. Nevertheless, recent
advances in molecular biology seems to suggest a profound synergy between these
two independent and biologically distinct molecular processes apparently
orchestrated by TFs.
CONFLICT OF INTERESTS
The Authors have no conflicts of interest to declare.
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Peer reviewer: Barry
Milavetz, PhD, Professor, Department of Basic Sciences, School of Medicine,
University of North Dakota, 501 Columbia Road, Grand Forks, ND 58203 USA;
ChorngHorng Lin, PhD, Associate Professor, Departent of Bioresources, DaYeh
University, 168 University Road, Da Cun, Changhwa, 515, Taiwan.
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