A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks
Michael Durando
1
Satoshi Tateishi
0
Cyrus Vaziri
1
0
Division of Cell Maintenance, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University
, Honjo 2-2-1, Kumamoto 860-0811,
Japan
1
Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill
,
Chapel Hill, NC 27599, USA
-
Trans-lesion DNA synthesis (TLS) is a DNA
damage-tolerance mechanism that uses low-fidelity
DNA polymerases to replicate damaged DNA. The
inherited cancer-propensity syndrome xeroderma
pigmentosum variant (XPV) results from error-prone TLS
of UV-damaged DNA. TLS is initiated when the Rad6/
Rad18 complex monoubiquitinates proliferating cell
nuclear antigen (PCNA), but the basis for recruitment
of Rad18 to PCNA is not completely understood. Here,
we show that Rad18 is targeted to PCNA by DNA
polymerase eta (Polg), the XPV gene product that is
mutated in XPV patients. The C-terminal domain of
Polg binds to both Rad18 and PCNA and promotes
PCNA monoubiquitination, a function unique to Polg
among Y-family TLS polymerases and dissociable
from its catalytic activity. Importantly, XPV cells
expressing full-length catalytically-inactive Polg exhibit
increased recruitment of other error-prone TLS
polymerases (Poli and Poli) after UV irradiation. These
results define a novel non-catalytic role for Polg in
promoting PCNA monoubiquitination and provide a
new potential mechanism for mutagenesis and
genome instability in XPV individuals.
Living organisms are constantly exposed to ubiquitous
genotoxins from endogenous and external sources (1).
However, cells have evolved numerous DNA damage
response (DDR) pathways that protect genomic DNA
and prevent genetic instability (2). Trans-lesion synthesis
(TLS) is a DDR mechanism involving specialized DNA
polymerases that can replicate damaged DNA templates (3).
TLS relies on inherently error-prone DNA polymerases of
the Y family to replicate damaged DNA (4). TLS by
Y-family polymerases (PolZ, Poli, Polk and Rev1) (5)
maintains replication in cells harbouring damaged DNA, albeit at
the cost of reduced fidelity. Each TLS polymerase performs
relatively error-free replication past a preferred cognate
lesion; in the absence of the appropriate TLS polymerase
for its preferred lesion, mutagenic replication by error-prone
polymerases predisposes to genetic instability (2).
PolZ is unique among Y-family polymerases in its
ability to perform accurate replication past UV-damaged
DNA (6,7). Lack of PolZ in the inherited
cancerpropensity syndrome xeroderma pigmentosum variant
(XPV) (8) results in error-prone replication by other
Y-family polymerases in sunlight-exposed cells (9,10).
Thus, UV-induced mutagenesis due to PolZ deficiency
compromises genetic integrity to manifest as exquisite
sunlight sensitivity and early skin cancer propensity.
A prerequisite for error-prone replication in TLS is the
Rad6/Rad18-mediated monoubiquitination of
proliferating cell nuclear antigen (PCNA) at the highly conserved
lysine K164 (11,12). Y-family polymerases contain
ubiquitin-binding (UBZ) domains that confer affinity to
monoubiquitinated PCNA (13,14). Failure to
monoubiquitinate PCNA at K164 phenocopies XPV by
compromising TLS and sensitizing cells to UV light and
other ubiquitous genotoxins (1518). Several other DDR
pathways also depend on PCNA monoubiquitination,
including SHPRH/HTLF-mediated template switching
(19), ZRANB3-dependent replication fork restart (20),
SNM1A-dependent intrastrand cross-link repair (21) and
the Fanconi Anaemia pathway activation (22).
Despite its pivotal role in the DDR, the molecular
mechanisms regulating Rad18-mediated PCNA
monoubiquitination are incompletely understood. The Rad18
Rad6 complex is thought to be recruited to the vicinity of
damaged DNA via direct interactions with RPA-coated
ssDNA (23,24). However, Rad18 lacks PCNA-binding
motifs, and it is unclear how Rad18 is targeted specifically
to PCNA at stalled forks (or other sites of post-replication
repair). A recent report by Zou and colleagues (25)
identified Spartan as a binding partner of both Rad18
and PCNA and proposed that Spartan acts as a scaffold
for recruiting Rad18 to PCNA. Consistent with a role for
Spartan in targeting Rad18 to PCNA, those workers
found DNA damage-induced PCNA monoubiquitination
was modestly attenuated in Spartan-depleted cells.
However, several other more recent publications have
reported alternative roles for Spartan in DNA damage
signalling (2629), and it is unclear whether Spartan or
alternative putative mediators exist to facilitate
recruitment of Rad18 to PCNA.
In mammalian cells, Rad18 exists in complex with PolZ
(30,31), and association of Rad18 with PolZ is necessary
for normal DNA damage tolerance (3032). Assembly of
the Rad18PolZ complex is stringently controlled by Cdc7
and Chk1 kinases, which serve to integrate TLS with
S-phase progression and the S-phase checkpoint,
respectively (30,32). Here we report that the PolZRad18
interaction plays a (...truncated)