Mechanisms for ensuring replisome processivity
The replisome includes separate polymerase machineries for the leading
and lagging strands, and the replicative helicase complex
(Cdc45-MCM-GINS or CMG) which removes obstacles and unwinds DNA in front
of the polymerases, as well as numerous auxiliary and regulatory factors
(reviewed in (Pellegrini, 2023)). In unperturbed replication, the
polymerase machineries are connected to the CMG to ensure that the
replisome travels as a unit and to allow regulation of fork progression
rate (Gambus et al., 2006; Jones et al., 2021; Simon et al., 2014).
When the replisome encounters an obstacle, either the CMG complex is
uncoupled and continues along the template while one or both of the
polymerases pause, or the whole replisome pauses. The former outcome
results in the characteristic signature of replication stress (Figure
1B) - an accumulation of single stranded DNA ahead of the replication
fork - observed with treatments that cause base lesions (eg: MMS) or
impair DNA polymerase activity (eg: HU). Persistent accumulation of
ssDNA around the replication fork is recognised by the kinase Mec1 (the
yeast homolog of ATR) via Ddc2 and the 9-1-1 complex, which then
phosphorylates histone H2A at S129 in neighbouring chromatin, forming a
focus of ɣH2A (Feng et al., 2006; Lopes et al., 2006; Namiki and Zou,
2006; Pardo et al., 2017; Puddu et al., 2011).
For base lesions such as 8-oxo-2’-deoxyguanosine (8-oxo-dG), the DNA
polymerases in the replisome are replaced with trans-lesion synthesis
enzymes Rad30 (Polη), Rev3/7 (Polζ) and/or Rev1, allowing DNA
replication to continue past the lesion while (hopefully) inserting the
correct base, with the lesion itself being left in place for NER or BER
to cure post-replication (reviewed in (Powers and Washington, 2018; Zhao
and Washington, 2017)) (Figure 1B). Alternatively, template switching
can be used to bypass lesions. In this process, the stalled polymerase
temporarily switches to using the other newly synthesised DNA strand as
a template to bypass obstacles (reviewed in (Ripley et al., 2020))
(Figure 1B). Both strategies aim to restart efficient DNA synthesis on
both strands at the replication fork, resulting in a functional
replication fork that is no longer associated with the replicative
helicase, an equivalent situation to a fork paused for example by a
short pulse of HU (Petermann et al., 2010). In both cases, replication
appears to resume as normal suggesting that these replication forks
re-couple to the CMG helicase, probably facilitated by the slower
progression of CMG that is uncoupled from the replicative polymerases
(Graham et al., 2017; Petermann et al., 2010).
The CMG helicase can traverse almost all replisome obstacles, and can
switch from single and double stranded binding modes when uncoupled and
back to single stranded binding once re-coupled(Wasserman et al., 2019).
However, programmed replication fork barriers can block CMG progression
resulting in a stable fork arrest, personified by the RFB in S.
cerevisiae ribosomal DNA and RTS1 at the S. pombemating-type locus (reviewed in (Labib and Hodgson, 2007)). This arrest
is mediated not by a physical block to the replisome but by the Fork
Protection Complex (FPC), which travels with the CMG complex (reviewed
in (Shyian and Shore, 2021)). Similarly, the DNA replication checkpoint,
which responds to global replication stress, can pause and stabilise
replication forks without uncoupling also through the FPC (Noguchi et
al., 2004; Pardo et al., 2017), though at least in human cells and
likely in yeast the forks become progressively less stable with time
(Petermann et al., 2010).
Only if bypass and restart mechanisms fail, and additionally the fork is
not resolved by an oncoming replication fork from another origin, must
further more potentially mutagenic options be explored. Once
disconnected from the CMG helicase, a stalled fork gains the capability
to reverse by re-annealing of the nascent leading and lagging strands to
yield a 4-pronged structure akin to a Holliday Junction and capable of
migration over significant distances, a remodelling process assisted by
Rad5 in budding yeast (Blastyak et al., 2007; Toth et al., 2022; Unk et
al., 2010) (Figure 1C). Reversed replication forks gain a free double
stranded end, which can be resected and initiate homologous
recombination (Cotta-Ramusino et al., 2005; Lemacon et al., 2017).
Homologous recombination can restart the replication process, albeit in
a different form known as Break-Induced Replication (BIR) in which the
leading strand is copied as a migrating D-loop, then the lagging strand
copied from the newly synthesised leading strand (Kramara et al., 2018;
Liu and Malkova, 2022; Malkova and Ira, 2013) (Figure 1C). Therefore,
whereas in canonical DNA replication both newly formed DNA helices
contain one strand from the original DNA template (known as ’semi
conservative replication’), in BIR both strands of the daughter DNA
helix are newly synthesised (’conservative replication’). BIR uses a
different DNA polymerase complement and relies on other helicases for
processivity (notably Pif1) (Kramara et al., 2018; Liu and Malkova,
2022; Lydeard et al., 2010); movement of this complex is slow and error
prone when forced to proceed over long distances, but in almost all
instances this problem is avoided as the BIR fork is resolved by a
replication fork coming from the other direction (Liu et al., 2021;
Mayle et al., 2015). Because BIR is innately slow and error prone,
replication forks are actively protected from resection so that
transient fork pausing can be overcome with a non-recombinational system
that likely does not involve a loss of the replisome structure or
function (Brambati et al., 2018). Single strand gaps are also
converted to double strand breaks when encountered by the replisome and
could enter a similar recombinational restart pathway (Vrtis et al.,
2021). However, this must again be a last resort, and mammalian cells
use PARP to guard against this outcome and it is speculated that yeast
use the FPC, at least for Top1-mediated damage (Ray Chaudhuri et al.,
2012; Westhorpe et al., 2020).
BIR forks have much more potential for introducing mutations than normal
replication forks (Deem et al., 2011; Pardo and Aguilera, 2012; Sakofsky
et al., 2014); the lack of the CMG complex makes these prone to further
stalling and recombinational repair if unresolved (Liu et al., 2021;
Smith et al., 2007). Furthermore, proper sister chromatid cohesion
requires replisome factors including the FPC, meaning that
recombinational repair during BIR is much more likely to result in
non-allelic homologous recombination (van Schie and de Lange, 2021).
Template switching based on microhomology is also more frequent,
resulting in complex structural variations showing multiple switches
with more or less homology at break points (Pardo and Aguilera, 2012;
Sakofsky et al., 2015). These properties make the slow progression of
BIR forks actively advantageous to the cell by providing the highest
chance of resolution by a normal replication fork coming from the other
direction and limiting the chances of mutation.
Therefore, stalled forks are protected from introducing further
mutations at multiple levels. Firstly, these forks are protected from
processing by the recombination machinery for as long as possible,
secondly the recombination events are tightly restrained and thirdly the
low processivity of BIR forks favours resolution by oncoming forks.