EXO1;  Exonuclease 1;  HEX1;  hEXO1

EXO1 is the only known nuclease, which is active in MMR in human cells. The MMR is a post-replicative DNA repair system that repairs certain damaged bases, base mismatches and base insertions/deletions, and various sizes of DNA loops (IDL). EXO1 has conserved domains and interacts directly with MMR factor MutS homolog 3 (MSH3) at the NH₂-terminus of EXO1 and with MutL homolog 1 (MLH1) and MSH2 at the COOH-terminus. During the replication process, in the S-phase of the cell cycle, the proteins EXO1, MSH2, and PCNA co-localize (Liberti et al. 2011). In humans, the MMR is carried out by the MutSα complex, a heterodimer of MSH2-MutS homolog 6 (MSH6), which recognizes mainly single-base mismatches or the MutSβ complex, a heterodimer of MSH2-MSH3, which recognizes IDL. The MutSα and β complexes operate by binding to the DNA lesion, followed by binding of the MutLα, a heterodimer of MLH1/postmeiotic segregation increased 2 (PMS2), which forms a ternary complex with the MutSα or Mutsβ at the base damage site. Next, PCNA and replication factor C (RFC) are recruited and stimulate the MutLα to nick the DNA by use of the intrinsic endonuclease activity in PMS2; these nicks are created both 3′ and 5′ to the lesion. Subsequently, EXO1 is recruited to the damaged site to excise the damaged base or during replication to remove the newly synthesized DNA containing the error in a MutSα or (β) and MutLα-dependent manner. Replication protein A (RPA) facilitates protection of the single-stranded DNA (ssDNA) intermediates during the repair process to prevent formation of secondary structures and DNA degradation. In addition, RPA is involved in regulating accessibility of EXO1 to the DNA, ensuring that excision is only allowed in the presence of a replication error. In a joint activity, PCNA and DNA polymerase δ and DNA polymerase ε resynthesize the DNA and DNA ligase I and finalize the process by ligation of the nick (Kunkel and Erie 2015).

Recent reports indicate that EXO1 is recruited to DNA damage in a PAR-mediated (poly(ADP-ribosyl)ation (PARylation)) manner (Zhang et al. 2015; Cheruiyot et al. 2015). PAR is a posttranslational modification, synthetized by Poly(ADP-ribose) polymerase 1 (PARP1), which provide a PAR chain docking platform on EXO1 for additional DNA damage repair factors which are rapidly recruited to the DNA damage site (Tallis et al. 2014; Cheruiyot et al. 2015). A PAR interaction motif, also named PIN motif, is located at the NH₂-terminus of EXO1 (Fig. 1) (Zhang et al. 2015; Cheruiyot et al. 2015). In vitro experiments demonstrated that PARP1 physically interacts with EXO1 and stimulates EXO1 in its 5′ excision activity an in vitro MMR assay (Liu et al. 2011). A PAR-binding motif (¹²⁵ITHAMAHKVIK¹³⁵) in EXO1 was predicted (Gagné et al. 2008), but mutation of two essential residues in the PAR-binding motif to alanine (¹²⁵ITHAMAAAVIA¹³⁵) failed to show a difference in recruitment of EXO1 to DNA damage (Cheruiyot et al. 2015). Zhang and colleagues showed that an EXO1 mutant, EXO1-R93G, abolished the interaction with PAR and recruitment in cells to dsDNA damage (Zhang et al. 2015). During this initial stage of damage association, EXO1 resection activity, 5′ exo, and 5′ flap activity are held inactive by PAR until its clearance by poly(ADP-ribose) glycohydrolase (PARG) and other repair factors including PCNA is recruited (Cheruiyot et al. 2015).

During the resection of DSBs, an interplay between the MRE11-RAD50-NBS1 (MRN) complex and EXO1 is well documented (Nimonkar et al. 2011). A recent study showed that EXO1 is essential for embryogenesis and the DNA damage response in mice Exo1 ^null/null with hypomorphic Nijmegen breaking syndrome 1 (Nbs1) allele (Rein et al. 2015). NBS1 is a partner in the MRN complex, and deletion of either of the genes in the MRN complex is lethal, while deletion of Exo1 has a minimal impact in cells or mice (Williams et al. 2002; Schaetzlein et al. 2013). Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 (Nbs1^ΔB/ΔB) leads to severe developmental impairment, embryonic death, and chromosomal instability (Rein et al. 2015). The combination of Nbs1^ΔB/ΔB-/EXO1-depleted cells are strongly affected in DNA replication, DNA repair, checkpoint signaling, and DNA damage response (Rein et al. 2015). Human EXO1 interacts directly with the RecQ helicases RECQL1, BLM, and WRN and is stimulated in its nuclease activity (Fig. 3) (reviewed in Keijzers et al. 2014). Additionally, EXO1 is regulated at the DNA ends by proteins such as WRN, RPA, Ku70/80, or CtIP ((C-terminal-binding protein) (CtBP)-interacting protein) to avoid unlimited resection (Eid et al. 2010; Iannascoli et al. 2015). Cells depleted in WRN and treated with DNA topoisomerase 1 (TOP1) inhibitor, camptothecin, which creates single-strand breaks (SSB) and eventually DSBs, show an enhanced degradation and ssDNA formation at nascent strands by the combined action of MRE11 and EXO1, as opposed to the limited processing of nascent strands performed by DNA2 in wild-type cells (Iannascoli et al. 2015). This suggests that WRN exonuclease activity prevents unscheduled degradation by MRE11 and EXO1 and thereby supports the restart of stalled forks and limits chromosome breakage (Iannascoli et al. 2015).

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It has been suggested that DNA resection occurs via two routes. Either via a RPA-BLM-DNA2-MRN mediated route, where the MRN complex promotes recruitment of BLM to DNA end and RPA stimulates BLM in DNA helices unwinding to stimulate 5′ → 3′ resection by DNA2 (Nimonkar et al. 2011). The other 5′ → 3′ resection route is mediated by EXO1 and stimulated by BLM, MRN, and RPA in DNA resection (Nimonkar et al. 2011). The Ku70/80 heterodimer, the center molecule in the DNA repair pathway nonhomologous end-joining (NHEJ), possesses a high affinity for double-stranded DNA ends. The Ku70/80 heterodimer prevents over-resection by nucleases from the DNA ends by binding tightly to the DNA ends. Studies in yeast showed that Ku70/80 blocks EXO1-mediated DNA end resection at forked dsDNA substrates. Another protein which modulates EXO1 activity is CtIP; it interacts directly with EXO1 and inhibits its exonuclease activity in vitro (Eid et al. 2010). Microhomology-mediated end joining (MMEJ) is an error-prone double stranded break DNA repair mechanism, which targets long stretches of single-stranded DNA up to 25 nucleotides, and is thought to be more mutagenic than the NHEJ. The MMEJ can function independently of the NHEJ factors (Ku70/80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ligase IV, and X-ray cross complementation group 4 protein (XRCC4)) and is active during G2-, S-, or G1-phase in the cell cycle. The MMEJ double-stranded DNA repair pathway includes BLM, MRN, EXO1, DNA2, FEN1, DNA polymerase β, λ, or μ, Ligase I or Ligase III/XRCC1, and the MMR proteins and has additional roles in humans in meiosis and class switching (see below). The MMEJ repair pathway is less well characterized than other DNA repair pathways; however the role of BLM stimulated by the exonuclease EXO1 or DNA2 during the MMEJ is well documented.

EXO1 activity plays an important role during meiosis. Mice studies show that both Exo1^Δ6/Δ6 (Exo1 deleted in exon 6) and Exo1 ^null/null male and female mice were infertile when mated with wild-type mice (Wei et al. 2003; Schaetzlein et al. 2013). The Exo1 ^null/null mice had smaller testes, but this reduction was not caused by a decreased body weight in adult males (Schaetzlein et al. 2013). The Exo1^Δ6/Δ6 as well as Exo1 ^null/null male mice were severely depleted of spermatids and spermatozoa (Wei et al. 2003; Schaetzlein et al. 2013). Further analysis of spermatogenesis in Exo1 ^null/null mice revealed that only a very small number of spermatogenic cells progress through meiosis II, which was indicated by the very few spermatozoa that could be retrieved from the epididymis of Exo1 ^null/null (Schaetzlein et al. 2013), suggesting that mouse EXO1 requires its catalytic activity, either endo- or exo-activity, to progress through meiosis II. The presence of pachytene spermatocytes in both Exo1^Δ6/Δ6 and Exo1 ^null/null male mice indicates that meiosis can progress through prophase I (Wei et al. 2003; Schaetzlein et al. 2013).

Diversity in antibodies occurs in two phases in B cells. In pre-B cells, rearrangement of variable (V), diversity (D), and joining (J) gene segments (V(D)J) at the immunoglobulin heavy chain region is initiated by the recombination-activating genes 1 and 2 (RAG1 and RAG2). Immature B cells will be preceded by class switch recombination (CSR) in the S (switch) region. The process of CSR is initiated by activation-induced deaminase (AID). AID creates mutations by substituting cytosine for uracil in the Ig loci at well-defined regions encoding rearranged V genes on the heavy and light chain loci and S regions on the heavy chain locus. Next, the BER pathway enzyme uracil-DNA glycosylase (UNG), in addition to MMR enzymes, facilitates the removal of mismatched bases leading to DSBs and deletions in the S region (rearrangement), creating increased variability. The created DSBs during gene segment rearrangement are repaired by the NHEJ as well as by MMEJ. B cells deficient in NHEJ require MMR proteins MLH1, MSH2, and EXO1 during CSR (Eccleston et al. 2011). The Exo1^Δ6/Δ6 mice predominantly developed lymphoma between 16 and 18 months compared to Exo1^+/Δ6 and wild-type mice (Wei et al. 2003). Additional studies in the Exo1^Δ6/Δ6 demonstrated that the EXO1-mutated mice have decreased CSR and alteration in SHM by normal AID functioning (Bardwell et al. 2004). Interestingly, the Exo1^Δ6/Δ6 phenotype suggested that mutation in the V region connects EXO1 and MLH1 to CSR and somatic hypermutation (SHM) (Bardwell et al. 2004). These observations are supported by Sμ tandem repeat mice (SμTR^−/− mice); when crossed with Mlh1 ^−/− or Exo1^Δ6/Δ6, an increased switch junction microhomology was noted compared to Msh2 ^−/− mice (Eccleston et al. 2009). The Exo1 ^null/null showed that the EXO1 nuclease activity has minor roles in correction of replication errors during MMR and in the generation of mutations by somatic hypermutation at Ig heavy chain region (Schaetzlein et al. 2013). B cells of the Exo1 ^null/null male mice, stimulated by either LPS or LPS/IL-4, failed to induce efficient CSR to switch from IgM to IgG3 or to IgG1, but this was not caused by impaired cell proliferation (Schaetzlein et al. 2013). These observations suggest that the MLH1 and EXO1 are dispensable in the early steps of CSR or in the promotion of AID- and MMR-triggered DSBs at the switch regions but required in microhomology-mediated resection joining the DNA ends.

Maintenance of the 3′ end of telomeres is essential for genome stability. Telomeres are packaged in loop structures in telomere loops (T-loops) and protected by the shelterin complex, including telomeric repeat factor 1 and telomeric repeat factor 2 (TFR1 and TRF2), protection of telomeres protein 1 (POT1), repressor activator protein 1 (RAP1), (TRF1)-interacting nuclear factor 2 (TIN2), and telomere protection protein (TPP1), and cooperating with accessory proteins such as MRE11, Ku70/80, and helicases BLM, WRN, and RECQL4. The 3′ telomere end invades the paired DNA at the telomeric region and forms a protective loop, thus forming a three-stranded DNA displacement loop (also called D-loop).

Studies in mice showed that during the cell cycle, the 3′ overhang synthesis is differently regulated at the leading- and lagging-end telomere, by the nucleases Apollo and EXO1, which initiates formation of the 3′ overhang at the leading- but not at the lagging-end telomeres. POT1 regulates Apollo at the telomere end to avoid hyper-resection; however extensive resection of the telomere ends generates transient long 3′ overhangs in the S/G2 cell cycle phase (Wu et al. 2012). Interestingly, telomere-dysfunctional mice Exo1^{Δ6/Δ6/mTerc−/−} deleted in EXO1 and telomerase RNA component (TERC) improve organ maintenance and life span (Schaetzlein et al. 2007).

EXO1 is a 5′ → 3′ exonuclease, a 5′ flap endonuclease, and a 5′ RNase activity (Lee and Wilson 1999; Qiu et al. 1999; Keijzers et al. 2015). EXO1 is generally expressed in most tissue. During replication EXO1 co-localizes with MMR repair protein MSH2 and cell cycle regulator PCNA (Liberti et al. 2011). The EXO1 protein participates in both MMR and DSB repair. Mice studies suggest roles of EXO1 in meiosis, specifically in the spermatogenesis development; however a contribution of EXO1 to human meiosis is unknown. In both humans and mice, EXO1 has important roles in the MMEJ repair pathway, which is essential in immunoglobulin development specifically in CSR. Studies in mice suggest that EXO1 contributes to the formation of the 3′ overhang at leading-end telomeres (Wu et al. 2012), but less is known on the roles of EXO1 in telomere maintenance in humans.