Pathway: Drug-induced formation of DNA interstrand crosslinks
Drug-induced formation of DNA interstrand crosslinks
FANCD2 and FANCI form a complex and are mutually dependent on one another for their respective monoubiquitination. After DNA damage and during S phase, FANCD2 localizes to discrete nuclear foci that colocalize with proteins involved in homologous recombination repair, such as BRCA1 and RAD51. The FA pathway is regulated by ubiquitination and phosphorylation of FANCD2 and FANCI. ATR-dependent phosphorylation of FANCI and FANCD2 promotes monoubiquitination of FANCD2, stimulating the FA pathway (Cohn and D'Andrea 2008, Wang 2007). The complex of USP1 and WDR48 (UAF1) is responsible for deubiquitination of FANCD2 and negatively regulates the FA pathway (Cohn et al. 2007).
Monoubiquitinated FANCD2 recruits DNA nucleases, including SLX4 (FANCP) and FAN1, which unhook the ICL from one of the two covalently linked DNA strands. The DNA polymerase nu (POLN) performs translesion DNA synthesis using the DNA strand with unhooked ICL as a template, thereby bypassing the unhooked ICL. The unhooked ICL is subsequently removed from the DNA via nucleotide excision repair (NER). Incision of the stalled replication fork during the unhooking step generates a double strand break (DSB). The DSB is repaired via homologous recombination repair (HRR) and involves the FA genes BRCA2 (FANCD1), PALB2 (FANCN) and BRIP1 (FANCJ) (reviewed by Deans and West 2011, Kottemann and Smogorzewska 2013). Homozygous mutations in BRCA2, PALB2 or BRIP1 result in Fanconi anemia, while heterozygous mutations in these genes predispose carriers to primarily breast and ovarian cancer. Well established functions of BRCA2, PALB2 and BRIP1 in DNA repair are BRCA1 dependent, but it is not yet clear whether there are additional roles for these proteins in the Fanconi anemia pathway that do not rely on BRCA1 (Evans and Longo 2014, Jiang and Greenberg 2015). Heterozygous BRCA1 mutations predispose carriers to breast and ovarian cancer with high penetrance. Complete loss of BRCA1 function is embryonic lethal. It has only recently been reported that a partial germline loss of BRCA1 function via mutations that diminish protein binding ability of the BRCT domain of BRCA1 result in a FA-like syndrome. BRCA1 has therefore been designated as the FANCS gene (Jiang and Greenberg 2015).
The FA pathway is involved in repairing DNA ICLs that arise by exposure to endogenous mutagens produced as by-products of normal cellular metabolism, such as aldehyde containing compounds. Disruption of the aldehyde dehydrogenase gene ALDH2 in FANCD2 deficient mice leads to severe developmental defects, early lethality and predisposition to leukemia. In addition to this, the double knockout mice are exceptionally sensitive to ethanol consumption, as ethanol metabolism results in accumulated levels of aldehydes (Langevin et al. 2011).
Depending on the type of DNA damage and the cell cycle status, the DNA repair machinery utilizes several different pathways to restore the genome to its original state. When the damage and circumstances are such that the DNA cannot be repaired with absolute fidelity, the DNA repair machinery attempts to minimize the harm and patch the insulted genome well enough to ensure cell viability.
Accumulation of DNA alterations that are the result of cumulative DNA damage and utilization of "last resort" low fidelity DNA repair mechanisms is associated with cellular senescence, aging, and cancer. In addition, germline mutations in DNA repair genes are the underlying cause of many familial cancer syndromes, such as Fanconi anemia, xeroderma pigmentosum, Nijmegen breakage syndrome and Lynch syndrome, to name a few.
When the level of DNA damage exceeds the capacity of the DNA repair machinery, apoptotic cell death ensues. Actively dividing cells have a very limited time available for DNA repair and are therefore particularly sensitive to DNA damaging agents. This is the main rationale for using DNA damaging chemotherapeutic drugs to kill rapidly replicating cancer cells.
There are seven main pathways employed in human DNA repair: DNA damage bypass, DNA damage reversal, base excision repair, nucleotide excision repair, mismatch repair, repair of double strand breaks and repair of interstrand crosslinks (Fanconi anemia pathway). DNA repair pathways are intimately associated with other cellular processes such as DNA replication, DNA recombination, cell cycle checkpoint arrest and apoptosis.
The DNA damage bypass pathway does not remove the damage, but instead allows translesion DNA synthesis (TLS) using a damaged template strand. Translesion synthesis allows cells to complete DNA replication, postponing the repair until cell division is finished. DNA polymerases that participate in translesion synthesis are error-prone, frequently introducing base substitutions and/or small insertions and deletions.
The DNA damage reversal pathway acts on a very narrow spectrum of damaging base modifications to remove modifying groups and restore DNA bases to their original state.
The base excision repair (BER) pathway involves a number of DNA glycosylases that cleave a vast array of damaged bases from the DNA sugar-phosphate backbone. DNA glycosylases produce a DNA strand with an abasic site. The abasic site is processed by DNA endonucleases, DNA polymerases and DNA ligases, the choice of which depends on the cell cycle stage, the identity of the participating DNA glycosylase and the presence of any additional damage. Base excision repair yields error-free DNA molecules.
Mismatch repair (MMR) proteins recognize mismatched base pairs or small insertion or deletion loops during DNA replication and correct erroneous base pairing by excising mismatched nucleotides exclusively from the nascent DNA strand, leaving the template strand intact.
Nucleotide excision repair pathway is involved in removal of bulky lesions that cause distortion of the DNA double helix. NER proteins excise the oligonucleotide that contains the lesion from the affected DNA strand, which is followed by gap-filling DNA synthesis and ligation of the repaired DNA molecule.
Double strand breaks (DSBs) in the DNA can be repaired via a highly accurate homologous recombination repair (HRR) pathway, or through error-prone nonhomologous end joining (NHEJ), single strand annealing (SSA) and microhomology-mediated end joining (MMEJ) pathways. DSBs can be directly generated by some DNA damaging agents, such as X-rays and reactive oxygen species (ROS). DSBs can also be intermediates of the Fanconi anemia pathway.
Interstrand crosslinking (ICL) agents damage the DNA by introducing covalent bonds between two DNA strands, which disables progression of the replication fork. The Fanconi anemia proteins repair the ICLs by unhooking them from one DNA strand. TLS enables the replication fork to bypass the unhooked ICL, resulting in two replicated DNA molecules, one of which contains a DSB and triggers double strand break repair, while the sister DNA molecule contains a bulky unhooked ICL, which is removed through NER.
Single strand breaks (SSBs) in the DNA, generated either by DNA damaging agents or as intermediates of DNA repair pathways such as BER, are converted into DSBs if the repair is not complete prior to DNA replication. Simultaneous inhibition of DSB repair and BER through cancer mutations and anti-cancer drugs, respectively, is synthetic lethal in at least some cancer settings, and is a promising new therapeutic strategy.
For reviews of DNA repair pathways, please refer to Lindahl and Wood 1999 and Curtin 2012.