Meta-Biol

• Pathways

MSH2:MSH3 (MutSbeta) binds unpaired loops of 2 or more nucleotides (Palombo et al. 1996, Genschel et al. 1998). Human cells contain about 6-fold more MSH2:MSH6 than MSH2:MSH3 (MutSbeta) and an imbalance in the ratio can cause a mutator phenotype (Drummond et al. 1997, Marra et al. 1998). Binding of the mismatch activates MSH2:MSH3 to exchange ADP for ATP, adopt the conformation to allow movement along the DNA, and interact with downstream effectors PCNA, MLH1:PMS2 and EXO1. The interaction with PCNA initiates excision of the recently replicated strand. MLH1:PMS2 makes a nick that is enlarged to a gap of hundreds of nucleotides by EXO1. DNA is polymerized across the gap by DNA polymerase delta and the remaining nick is sealed by DNA ligase I.

The mismatch repair (MMR) system corrects single base mismatches and small insertion and deletion loops (IDLs) of unpaired bases. MMR is primarily associated with DNA replication and is highly conserved across prokaryotes and eukaryotes. MMR consists of the following basic steps: a sensor (MutS homologue) detects a mismatch or IDL, the sensor activates a set of proteins (a MutL homologue and an exonuclease) that select the nascent DNA strand to be repaired, nick the strand, exonucleolytically remove a region of nucleotides containing the mismatch, and finally a DNA polymerase resynthesizes the strand and a ligase seals the remaining nick (reviewed in Kolodner and Marsischkny 1999, Iyer et al. 2006, Li 2008, Fukui 2010, Jiricny 2013).
Humans have 2 different MutS complexes. The MSH2:MSH6 heterodimer (MutSalpha) recognizes single base mismatches and small loops of one or two unpaired bases. The MSH2:MSH3 heterodimer (MutSbeta) recognizes loops of two or more unpaired bases. Upon binding a mismatch, the MutS complex becomes activated in an ATP-dependent manner allowing for subsequent downstream interactions and movement on the DNA substrate. (There are two mechanisms proposed: a sliding clamp and a switch diffusion model.) Though the order of steps and structural details are not fully known, the activated MutS complex interacts with MLH1:PMS2 (MutLalpha) and PCNA, the sliding clamp present at replication foci. The role of PCNA is multifaceted as it may act as a processivity factor in recruiting MMR proteins to replicating DNA, interact with MLH1:PMS2 and Exonuclease 1 (EXO1) to initiate excision of the recently replicated strand and direct DNA polymerase delta to initiate replacement of bases. MLH1:PMS2 makes an incision in the strand to be repaired and EXO1 extends the incision to make a single-stranded gap of up to 1 kb that removes the mismatched base(s). (Based on assays of purified human proteins, there is also a variant of the mismatch repair pathway that does not require EXO1, however the mechanism is not clear. EXO1 is almost always required, it is possible that the exonuclease activity of DNA polymerase delta may compensate in some situations and it has been proposed that other endonucleases may perform redundant functions in the absence of EXO1.) RPA binds the single-stranded region and a new strand is synthesized across the gap by DNA polymerase delta. The remaining nick is sealed by DNA ligase I (LIG1).
Concentrations of MMR proteins MSH2:MSH6 and MLH1:PMS2 increase in human cells during S phase and are at their highest level and activity during this phase of the cell cycle (Edelbrock et al. 2009). Defects in MSH2, MSH6, MLH1, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch syndrome) (reviewed in Martin-Lopez and Fishel 2013).

DNA repair is a phenomenal multi-enzyme, multi-pathway system required to ensure the integrity of the cellular genome. Living organisms are constantly exposed to harmful metabolic by-products, environmental chemicals and radiation that damage their DNA, thus corrupting genetic information. In addition, normal cellular pH and temperature create conditions that are hostile to the integrity of DNA and its nucleotide components. DNA damage can also arise as a consequence of spontaneous errors during DNA replication. The DNA repair machinery continuously scans the genome and maintains genome integrity by removing or mending any detected damage.

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.