P13 - investigated whether loss of dna repair functions or increased in dna damage alters cytokine responses and cell prolif

Tutored assignment of the IP Introductory course April 2006
Noordwijkerhout
Summary report of poster session CIII on
Fanconi Anemia and Crosslink repair
Silvia Costantini, Chris Dinant, Karin Garten and Natasha Iles
tutored by Federica Marini and Johan de Winter
Fanconi anemia (FA) is a rare, recessive chromosomal-instability disorder characterised by a striking hypersensitivity to agents that induce DNA interstrand crosslinks (ICLs). The disease is clinically highly heterogeneous, even between monozygotic twins (Auerbach and Allen, 1991; Kook, 2005) which dramatically complicates diagnosis. A hallmark feature of FA is pancytopenia due to increased apoptosis of hematopoietic cells. The genomic instability of the surviving cells leads to myelodysplastic syndrome and acute myeloid leukaemia, and the risk of developing solid tumours is also increased (Kook, 2005). Complications of bone marrow failure are the most common cause of death (Alter et al., 1991; Kook, 2005). The hypersensitivity of cells from FA patients to DNA ICL agents such as mitomycin C (MMC) is the basis for the diagnosis of FA. This type of damage prevents strand separation which is required for vital cellular processes such as DNA replication and transcription, and as a result are extremely cytotoxic especially in proliferating cells. Replication of DNA containing ICLs induces double strand breaks (DSBs) (De Silva et al., 2000; Niedernhofer et al., 2004). ICLs are also potent inducers of sister chromatid exchange (SCEs) which result from homologous recombination (HR) (Sonoda et al., 1999) most likely due to a collapsed replication fork (Helleday, 2003). This implies a role for DNA replication, replication-induced DSBs and subsequent repair of these DSBs by HR in the repair of ICLs. Increased levels of radial structures of chromosomes are also observed in FA cells treated with a crosslinking agent (Kook, 2005). These are created when broken sister chromatids of disparate chromosomes become erroneously fused to one another creating a tandem array of chromosomes connected via recombined sister chromatids. These result from failed repair by HR. In summary, it is likely that FA cells are defective in the HR-mediated repair of DSBs induced by ICLs. It is also possible that there may be a cell cycle checkpoint defect in FA cells resulting in failure to halt DNA replication to permit ICL repair (Sala-Trepat et al., 2000). The phenotypic heterogeneity of FA prompted a search for multiple genetic causes that could be responsible for the extreme clinical variation. The sensitivity of FA cells to ICLs allowed the identification of distinct complementation groups, where each complementation group represents FA patients with defects in the same gene. Such complementation groups can be identified when cells from different patients are fused together and the resulting cell population is screened for wild-type resistance to ICL damage by the complementation of each other’s defect. To date, the total number of FA genes is twelve (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L and -M). FANCD1 is identical to BRCA2 which is implicated in HR (Howlett et al., 2002), FANCL encodes an E3 ubiquitin ligase which is implicated in the monoubiquitination of FANCD2 – an essential step in the FA pathway (Meetei et al., 2003a), and FANCJ and FANCM encode DNA helicases (Meetei et al., 2005). Many of the FA proteins physically interact in the cell to form an FA core complex which includes FANCA, -B, -C, -E, -F, -G, -L and -M (Garcia-Higuera et al., 2001; Meetei et al., 2003a; Meetei et al., 2004; Meetei et al., 2005). In normal S phase and after treatment with ICL-inducing agents, following the probable ATR phosphorylation of FANCD2 (Pichierri and Rosselli, 2004), FANCL monoubiquitinates FANCD2, a process which requires FANCI. This triggers the association of FANCD2 with chromatin and its accumulation in nuclear foci. Activated FANCD2 colocalises with factors involved in HR-mediated DSB repair such as BRCA1, BRCA2/FANCD1 and RAD51 (Taniguchi et al., 2002). Replication factors such as PCNA and RPA are also present in these foci. The FA core complex is required for the translocation of FANCD2 to these foci and for the foci to restore replication and cell cycle progression (Matsushita et al., 2005). The FA core complex also interacts with the BLM-TOPOIIIα complex (Meetei et al., 2003b). This complex is involved in the proper dissolution of double
Holliday junctions which are intermediates of HR (Wu et al., 2005).
There are still many unanswered questions about FA and crosslink repair, and there are FA
proteins still awaiting identification. The work on this topic which was presented as posters in
Noordwijkerhout in April 2006 is summarised below.
Model Systems used to study Fanconi Anemia and Crosslink repair

The posters presented illustrated the range of model systems which can be used to study FA
and crosslink repair. One example is the use of human cells from FA patients as described in
a poster by de Vries et al.. Levitus et al. recently discovered the BRIP1/BACH1 helicase to be
the gene responsible for the defect in the FA complementation group J. In 7 out of 8 FA-J
patients, de Vries et al. found pathogenic mutations, either in the coding regions or in the
splice sites surrounding exon 3 or exon 17. In the eighth patient, who was identified as FA-J
by cell fusion and by the ability to monoubiquitinate FANCD2, only one heterozygous
pathogenic mutation could be detected. However, further investigation of the paternal allele
revealed an alternative exon arising from a deep intronic mutation which created a new,
strong donor splice site. In addition, data from de Vries et al. suggested that they have
discovered a new complementation group downstream of FANCD2. In candidate J cells, they
have shown the presence of FANCJ with no pathogenic mutations and also full-length
FANCD1/BRCA2, NBS1 and monoubiquitinated FANCD2. Cell fusion and sequencing
experiments are being employed to confirm the cells do not belong to complementation group
D1. If confirmed, this group represents - along with FA-J and FA-D1 - a third complementation
group with a defect in the FA pathway that is downstream of the monoubiquitination of
FANCD2.
Kluzek et al. described three Chinese hamster cell mutants belonging to one
complementation group that is defective in the FANCC gene. Compared to wild-type FANCC
cDNA, these mutants possess a 177bp deletion which results in a shortened FANCC protein.
FANCC knock-out cells are defective in the mono-ubiquitination of FANCD2, show normal
formation of nuclear RAD51 foci in response to DNA damage and display higher levels of
spontaneous and MMC induced chromosomal aberrations. The three mutants described
show a different phenotype, and are therefore being used as a tool to elucidate the FANCC
function in the cellular response to mutagenic treatment.
Wilson, Jones, Wang et al. also reported the characterisation of a Chinese hamster mutant,
termed DEB1. DEB1 closely resembles human Fanconi anemia (FA) cell lines in phenotype.
The FA core complex fails to form in DEB1 and the mutant also fails to express the
monoubiquitinated form of the FANCD2 protein. DEB1 has been assigned to the same
genetic complementation group as the previously published mutant V-H4. Like DEB1, V-H4
fails to express the FANCL and FANCC proteins and its sensitivity to MMC is corrected upon
transfection of the human FANCL cDNA. The data presented suggests, at least in hamster
cells, that the FANCL protein is important for expression of FANCC.
Due to a high rate of recombination, the chicken B cell line DT40 is often used as a system in
which targeted genes are knocked-out, and Arakawa et al. have utilised this system to study
RDM1. The RDM1 gene (for RAD52 Motif1) is a novel gene involved in the cellular response
to cisplatin in vertebrates. Ablation of RDM1 in DT40 cells resulted in a more than 3-fold
increase in sensitivity to cisplatin. However, NER was still functional in the RDM1 -/- cells.
RDM1 binds RNA and both ss and ds DNA, and it may be able to distinguish between the two
nucleic acids. RDM1 recognises DNA distortions induced by cisplatin-DNA adducts in vitro.
RDM1 may play a dynamic role in the context of chromatin, possibly as a DNA damage
recognition factor.
Model systems can also be used to identify novel genes and proteins involved in the DNA
crosslink repair pathway. Van Haaften et al. performed a comprehensive screen for the genes
that protect cells against ionizing radiation in C. elegans. Using genome-wide RNAi they
identified 45 genes required for response to IR. Knockdowns of 11 genes had impaired
radiation-induced cell cycle arrest and 7 genes are essential for apoptosis upon exposure to
irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA
damaging cancer drugs cisplatin, camptothecin and methyl methanesulphonate (MMS).
Almost all genes uncovered in the screen are conserved across animal phylogeny and their
relevance for humans was demonstrated by the radiation sensitivity of the knockdown human
cells.
Mechanistic Insight into Fanconi Anemia

Most FA proteins are involved in forming a nuclear multiprotein core complex that is required
for the activation of FANCD2 by monoubiquitination through FANCL. Only FANCJ/BRIP1 and
FANCD1/BRCA2 function downstream of the central FANCD2 protein as cells from both
groups are capable of monoubiquitinating FANCD2. Godthelp et al. have been studying the
cellular phenotype of FANCJ/BRIP1 and FANCD1/BRCA2 deficient cells. These cells are
equally sensitive to MMC, MMS and bleomycin, and both are not sensitive to X-ray irradiation.
However, FANCD1/BRCA2 deficient cells but not FANCJ/BRIP1 deficient cells have an
increased sensitivity to UV irradiation indicating that FANCD1/BRCA2 may play a role in the
recognition/or repair of UV-induced DNA damage. FANCD1/BRCA2 deficient cells are also
impaired in DNA damage-induced RAD51 nuclear foci formation whereas FANCJ/BRIP1
deficient cells form RAD51 foci with normal kinetics. As with all FA cells, persisting γH2AX foci
are observed after MMC in both the FANCJ/BRIP1 and FANCD1/BRCA2 deficient cells
indicating slower repair kinetics of double strand breaks than wild-type cells. Therefore, the
impaired RAD51 foci formation and the difference in UV sensitivity allows the
FANCD1/BRCA2 deficient cells to be distinguished from the FANCJ/BRIP1 deficient cells.
These results suggest that downstream FA-pathway components FANCJ/BRIP1 and
FANCD1/BRCA2 are part of the integrated FA/BRCA DNA damage response pathway or,
alternatively, that they represent sub-pathways that might be distinct from the FA pathway in
which only FANCD1/BRCA2 is directly connected to the process of homologous
recombination.
The mechanism by which the FA pathway is activated in response to stalled or collapsed
replication forks remains unclear. Following two different approaches, Grossi et al. and
Lyakhovich et al. proposed two models for the FANCD2 recruitment and role. Grossi et al.
analysed the nuclear distribution of FANCD2 and of the replication factories in cells exposed
to hydroxyurea. They found that as a consequence of a prolonged exposure to hydroxyurea,
FANCD2 is recruited within nuclear regions actively engaged in DNA replication. This
observation is consistent with other studies which suggest that FANCD2 is mobilized at the
stalled forks. However, after removal of the replication blocking agent, DNA synthesis in FA-A
cells continue unimpeded with replication units established before the hydoxyurea block.
Moreover, FANCD2 foci persist throughout S phase, long after the removal of hydroxyurea.
These foci do not colocalize with replication factories, but with nuclear regions enriched in
single stranded-DNA intermediates. Therefore, they concluded that FANCD2 is not involved
in the restart of stalled forks and they hypothesised that FA proteins may contribute to the
repair of postreplication gaps. Lyakhovich et al. analysed the relocation of FANCD2 to the
damaged sites at different time intervals in different cell types after UV irradiation. They found
that FANCD2 dynamically relocates to UV induced stalled replication forks and that H2AX
phosphorylation is required for chromatin binding of FANCD2 but is dispensable for FANCD2
monoubiquitination upon DNA damage. They also determined that the interaction between
FANCD2 and H2AX is BRCA1 dependent. Moreover, they found that FANCD2 and H2AX
cooperate in the same epistatic pathway in response to mitomycin C damage. Consequently
they suggested that an H2AX-mediated chromatin response is functionally connected to the
FA/BRCA pathway to prevent chromosome instability.
Wilson, Jones, Kupfer et al. reported that FANCG, via its tetratricopeptide repeat (TPR)
motifs, participates in forming several distinct protein complexes, including the FA core
complex and a second complex that contains FANCD2 and the known homologous
recombinational repair proteins BRCA2 and RAD51 paralog XRCC3. Furthermore, the N-
terminal phosphorylation of FANCG is important for the formation of the complex containing
BRCA2/FANCD2 and XRCC3.

Pancytopenia and bone marrow failure are features of FA. Oldenampsen et al. investigated
whether loss of DNA repair functions or increased in DNA damage alters cytokine responses
and cell proliferation in erythroid progenitors. Erythropoietin (Epo) is the main growth and
survival factor of erythroid progenitors. Epo levels are mostly high in FA patients suggesting a
poor response to Epo and decreased erythropoiesis. Erythroid progenitors from mice deficient
in FANCA, FANCG or ERCC1 (a nucleotide exchange protein that is required to excise DNA
crosslinks downstream of FA pathway) show a decreased epo-induced phosphorylation of
stat5 and erk1/2 and control of epo-regulated gene expression is attenuated. Strikingly, the
defective signalling only developed upon culture at atmospheric oxygen. Crosslinks induced
by MMC and inhibition of DNA synthesis by cytosine arabinoside (AraC) cause stalled
replication forks which resulted in impaired Epo-signalling in wild-type murine erythroid
progenitors in a p53-dependent manner. Erythroid progenitors lacking ERCC1, or treated with
MMC or AraC that impairs Epo-induced signalling, do not die but expansion is abrogated and
terminal differentiation is enhanced. Impaired signal transduction may be part of a general
p53-dependent senescence-like program of erythroid progenitors in response to DNA
damage, which may contribute to Epo-resistant anemia.
Novel proteins in Crosslink Repair

Repair of ICLs requires strand incisions to separate the two covalently attached strands of
DNA. Hanada et al. reported that Mus81-Eme1 can function as one of the required
endonucleases to resolve an ICL into a DNA double-strand break. A functional interaction
between Rad54 and Mus81 was also found suggesting a role for Mus81 and Rad54 in the
same replication dependent ICL repair pathway.
Mus308, a D.melanogaster protein with family A polymerase and DNA/RNA helicase
domains, is known to be involved in ICL sensitivity. Marini et al. characterised mammalian
POLQ, HEL308 and POLN which are all homologues of Mus308 and evidence suggested that
POLN is also involved in ICL repair. These homologues were also characterised using RNAi
technology in C.elegans.
A functional connection between the cohesin protein SMC1 and the FA pathway was
described by Bogliolo et al. SMC1 is involved in sister-chromatid cohesion after replication
and has now been shown to also have a role in monoubiquitination and localisation of
FANCD2 upon DNA damage induction. Furthermore, reduction of SMC1 expression leads to
hypersensitivity to ICLs.
Repair of Protein-DNA Crosslinks

A novel methodology for the isolation of DNA-protein crosslinks (DPCs) from human and
hamster cells has been developed and validated by Barker et al. This method was used for
the identification of a number of proteins crosslinked to DNA by clinically relevant doses of IR.
DPC removal was observed to be limited in repair-deficient cell lines, suggesting that the HR
pathway may be involved in their repair. The role of the nucleotide excision repair (NER)
pathway in DPC removal is less clear.
Protein-DNA crosslinks can occur by many mechanisms. Meiotic recombination is initiated by
a DSB which is repaired using the homologous chromosome as a template. This meiosis-
specific DSB is created by Rec12 through a non-reversible topoisomerase-like mechanism.
The covalently bound Rec12 has to be removed from the DNA to allow subsequent DSB end
resection and meiotic recombination. Evidence of an involvement of the S.pombe MRN
complex (Rad32/Rad50/Nbs1) in this removal was presented by Hartsuiker et al.. The data
indicated that the Rad32 (endo)nuclease activity is responsible for the Rec12 removal, and
strongly suggest that the MRN complex is also directly involved in Top1 and Top2 removal.

References

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