DNA polymerase theta (POLQ) extends annealed microhomologous 3'-ssDNA overhangs at DNA double strand breaks (DSBs), using opposing overhangs as templates. POLQ can perform strand displacement synthesis, extending the overhangs beyond ssDNA-dsDNA junction point, which leads to the formation of displaced strand flaps (Kent et al. 2015). PARP1 (or possibly PARP2) is necessary for the recruitment of POLQ to DNA DSBs. POLQ-mediated DNA synthesis during microhomology mediated end joining (MMEJ) (also known as alternative nonhomologous end joining or alt-NHEJ) counteracts homologous recombination repair (HRR) and promotes survival of cells with a compromised HR pathway (Mateos-Gomez et al. 2015). POLQ is error-prone and introduces single nucleotide substitutions during DNA synthesis. HRR-deficient epithelial ovarian cancers frequently overexpress POLQ, which correlates with an increased frequency of somatic point mutations in these tumors (Ceccaldi et al. 2015).

Flap endonuclease FEN1, DNA polymerase theta (POLQ) and PARP1 or PARP2 homo- or heterodimers are recruited to DNA double strand breaks (DSBs) resected by MRN and RBBP8 (CtIP) in the process of microhomology-mediated end joining (MMEJ). The mechanism of recruitment of FEN1, PARP1 (or PARP2) and POLQ, which are all necessary for MMEJ progression (Liang et al. 2005, Mansour et al. 2010, Sharma et al. 2015, Mateos-Gomez et al. 2015, Ceccaldi et al. 2015, Kent et al. 2015), is poorly defined. PARP1 (or PARP2) recognizes ssDNA. In the DNA polymerase beta (POLB)-dependent long patch base excision repair (BER), PARPs form ternary complexes with FEN1 and POLB (Prasad et al. 2001, Lavrik et al. 2001, Cistulli et al. 2004), and it is possible that a similar mechanism involving PARPs, FEN1 and POLQ operates in MMEJ. POLQ functions as a homodimer and facilitates annealing of two 3'-ssDNA overhangs through their microhomology regions. POLQ requires <20 nucleotide (nt) long resected overhangs (Kent et al. 2015). Microhomology regions are optimally 10-19 nt long (Sharma et al. 2015), and the annealing is facilitated if the microhomology region is GC-rich (Kent et al. 2015).

DNA polymerase theta (POLQ) performs strand displacement DNA synthesis during microhomology-mediated end joining (MMEJ) (Kent et al. 2015), which is expected to result in the formation of displaced 5'-ssDNA flaps. FEN1, a 5'-flap endonuclease, is a necessary participant of MMEJ (Liang et al. 2005, Sharma et al. 2015). By analogy with base excision repair (Klungland and Lindahl 1997, Liu et al. 2005), FEN1 is thought to cleave the 5'-flaps generated by POLQ-mediated DNA strand displacement synthesis during MMEJ, thus enabling the subsequent ligation step.

The complex of DNA ligase 3 (LIG3) and XRCC1 is necessary for the completion of microhomology-mediated end joining (MMEJ), although DNA ligase 1 (LIG1) may also be involved (Sharma et al. 2015). LIG3:XRCC1 is recruited to MMEJ sites by the MRN complex and ligates single strand nicks that remain after reparative DNA synthesis by DNA polymerase theta (POLQ) at DNA double strand break (DSB) sites (Della-Maria et al. 2011). The annealing of microhomology regions between two 3'-ssDNA overhangs of resected DNA DSBs during MMEJ leads to deletion of the intervening DNA sequence and one of the microhomology regions in repaired double strand DNA (dsDNA) (Ghezraoui et al. 2014). In addition, as POLQ is error-prone, repaired DNA contains base substitutions (Ceccaldi et al. 2015). Similar to nonhomologous end joining (NHEJ), MMEJ (also known as alternative-NHEJ) can also produce translocations by joining unrelated DNA molecules (Ghezraoui et al. 2014).

Homology directed repair (HDR) through microhomology-mediated end joining (MMEJ) is an error prone process also known as alternative nonhomologous end joining (alt-NHEJ), although it does not involve proteins that participate in the classical NHEJ. Contrary to the classical NHEJ and other HDR pathways, homologous recombination repair (HRR) and single strand annealing (SSA), MMEJ does not require ATM activation. In fact, ATM activation inhibits MMEJ. Therefore, MMEJ may be triggered when the amount of DNA double strand breaks (DSBs) overwhelms DNA repair machinery of higher fidelity or when cells are deficient in components of high fidelity DNA repair.

MMEJ is initiated by a limited resection of DNA DSB ends by the MRN complex (MRE11A:RAD50:NBN) and RBBP8 (CtIP), in the absence of CDK2-mediated RBBP8 phosphorylation and related BRCA1:BARD1 recruitment (Yun and Hiom 2009). Single strand DNA (ssDNA) at resected DNA DSB ends recruits PARP1 or PARP2 homo- or heterodimers, together with DNA polymerase theta (POLQ) and FEN1 5'-flap endonuclease. In a poorly studied sequence of events, POLQ promotes the annealing of two 3'-ssDNA overhangs through microhomologous regions that are optimally 10-19 nucleotides long. Using analogy with POLB-mediated long patch base excision repair (BER), it is plausible that PARP1 (or PARP2) dimers coordinate the extension of annealed 3'-ssDNA overhangs via POLQ-mediated strand displacement synthesis with FEN1-mediated cleavage of the resulting 5'-flaps (Liang et al. 2005, Mansour et al. 2011, Sharma et al. 2015, Kent et al. 2015, Ciccaldi et al. 2015, Mateos-Gomez et al. 2015). The MRN complex subsequently recruits DNA ligase 3 (LIG3) bound to XRCC1 (LIG3:XRCC1) to ligate the remaining single strand nicks (SSBs) at MMEJ sites (Della-Maria et al. 2011).

Similar to single strand annealing (SSA), MMEJ leads to deletion of one of the microhomology regions used for annealing and the DNA sequence in between two annealed microhomology regions. MMEJ, just like classical NHEJ, can result in genomic translocations (Ghezraoui et al. 2014). In addition, since POLQ is an error-prone DNA polymerase, MMEJ introduces frequent base substitutions (Ceccaldi et al. 2015).

Homology directed repair (HDR) of DNA double strand breaks (DSBs) requires resection of DNA DSB ends. Resection creates 3'-ssDNA overhangs which then anneal with a homologous DNA sequence. This homologous sequence can then be used as a template for DNA repair synthesis that bridges the DSB. HDR preferably occurs through the error-free homologous recombination repair (HRR), but can also occur through the error-prone single strand annealing (SSA), or the least accurate microhomology-mediated end joining (MMEJ).

HRR and SSA share the initial steps that involve ATM signaling, formation of the so-called ionizing radiation-induced foci (IRIF), extensive resection of DNA DSB ends and activation of ATR signaling. In homologous recombination, 3'-ssDNA overhangs anneal with complementary sister chromatid strands. In SSA, 3'-ssDNA overhangs anneal with each other through homologous direct repeats contained in each overhang, resulting in deletions of one of the repeats and the DNA sequence in between the repeats during DNA repair synthesis.

Contrary to HRR and SSA, which both involve annealing of long stretches of highly homologous DNA sequences, MMEJ entails annealing of short regions of two 3'-ssDNA overhangs (up to 20 nucleotides) and is therefore more promiscuous and more likely to join unrelated DNA molecules. The error rate of MMEJ is additionally increased by the low fidelity of the DNA polymerase theta (POLQ), which performs DNA repair synthesis in MMEJ.

For reviews of this topic, please refer to Khanna 2001, Thompson and Schild 2001, Thompson and Schild 2002, Thompson and Limoli 2003, Ciccia and Elledge 2010.