DNA repair protein XRCC4 (hXRCC4) also known as X-ray repair cross-complementing protein 4 is a protein that in humans is encoded by the XRCC4 gene. XRCC4 is also expressed in many other animals, fungi and plants. hXRCC4 is one of several core proteins involved in the non-homologous end joining (NHEJ) pathway to repair DNA double strand breaks (DSBs).

NHEJ requires two main components to achieve successful completion. The first component is the cooperative binding and phosphorylation of artemis by the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). Artemis cleaves the ends of damaged DNA to prepare it for ligation. The second component involves the bridging of DNA to DNA ligase 4, by hXRCC4, with the aid of Cernunnos-XLF. DNA-PKcs and hXRCC4 are anchored to Ku70 / Ku80 heterodimer, which are bound to the DNA ends.

Since hXRCC4 is the key protein that enables interaction of DNA ligase 4 to damaged DNA and therefore ligation of the ends, mutations in the XRCC4 gene were found to cause embryonic lethality in mice and developmental inhibition and immunodeficiency in humans. Furthermore, certain mutations in XRCC4 are associated with an increased risk of cancer.

Double strand breaks

Double strand breaks (DSBs) are mainly caused by free radicals generated from ionizing radiation in the environment and from by-products released continually during cellular metabolism. DSBs that are not efficiently repaired may result in the loss of important protein coding genes and regulatory sequences required for gene expression necessary for the life of a cell. DSBs that cannot rely on a newly copied sister chromosome generated by DNA replication to fill in the gap will go into the NHEJ pathway. This method of repair is essential as it is a last resort to prevent loss of long stretches of the chromosome. NHEJ is also used to repair DSBs generated during V(D)J recombination when gene regions are rearranged to create the unique antigen binding sites of antibodies and T-cell receptors.

Sources of DNA damage

DNA damage occurs very frequently and is generated from exposure to a variety of both exogenous and endogenous genotoxic sources. One of these include ionizing radiation, such as gamma radiation and X-rays, which ionize the deoxyribose groups in the DNA backbone and can induce DSBs. Reactive oxygen species (ROS), such as superoxide (O2–), hydrogen peroxide (H2O2), hydroxyl radicals (HO•), and singlet oxygen (1O2), can also produce DSBs as a result of ionizing radiation as well as cellular metabolic processes that are naturally occurring. DSBs can also be caused by the action of DNA polymerase while attempting to replicate DNA over a nick that was introduced as a result of DNA damage.

Consequences of DSBs

There are many types of DNA damage, but DSBs, in particular, are the most harmful as both strands are completely disjointed from the rest of the chromosome. If an efficient repair mechanism does not exist, the ends of the DNA can eventually degrade, leading to a permanent loss of sequence. A double-stranded gap in DNA will also prevent replication from proceeding, resulting in an incomplete copy of that specific chromosome, targeting the cell for apoptosis. As with all DNA damage, DSBs can introduce new mutations that can ultimately lead to cancer.

DSB repair methods

There are two methods for repairing DSBs depending on when the damage occurs during mitosis. If the DSB occurs after DNA replication has completed proceeding S phase of the cell cycle, the DSB repair pathway will use homologous recombination by pairing with the newly synthesized daughter strand to repair the break. However, if the DSB is generated prior to synthesis of the sister chromosome, then the template sequence that is required will be absent. For this circumstance, the NHEJ pathway provides a solution for repairing the break and is the main system used to repair DSBs in humans and multicellular eukaryotes. During NHEJ, very short stretches of complementary DNA, one base pair or more at a time, are hybridized together, and the overhangs are removed. As a result, this specific region of the genome is permanently lost and the deletion can lead to cancer and premature aging.

Properties

Gene and protein

XRCC4 is located on chromosome 5, specifically at 5q14.2. This gene contains eight exons and three mRNA transcript variants, which encode two different protein isoforms. Transcript variant 1, mRNA, RefSeq NM_003401.3, is 1688 bp long and is the shortest out of the three variants. It is missing a short sequence in the 3' coding region as compared to variant 2. Isoform 1 contains 334 amino acids. Transcript variant 2, mRNA, RefSeq NM_022406, is 1694 bp long and encodes the longest isoform 2, which contains 336 amino acids. Transcript variant 3, RefSeq NM_022550.2, is 1735 bp and is the longest, but it also encodes for the same isoform 1 as variant 1. It contains an additional sequence in the 5'UTR of the mRNA transcript and lacks a short sequence in the 3'coding region as compared to variant 2.

Structure

hXRCC4 is a tetramer that resembles the shape of a dumbbell containing two globular ends separated by a long, thin stalk. The tetramer is composed of two dimers, and each dimer is made up of two similar subunits. The first subunit (L) contains amino acid residues 1–203 and has a longer stalk than the second subunit (S) which contains residues 1–178.

The globular N-terminal domains of each subunit are identical. They are made up of two, antiparallel beta sheets that face each other in a beta sandwich-like structure (i.e., a "flattened" beta barrel) and are separated by two alpha helices on one side. The N-terminus begins with one beta sheet composed of strands 1, 2, 3, and 4, followed by a helix-turn-helix motif of the two alpha helices, αA and αB, which continues into strands 5, 6, 7, and ending with one alpha-helical stalk at the C-terminus. αA and αB are perpendicular to one another, and because one end of αB is partially inserted between the two beta sheets, it causes them to flare out away from each other. The beta sandwich structure is held together through three hydrogen bonds between antiparallel strands 4 and 7 and one hydrogen bond between strands 1 and 5.

The two helical stalks between subunits L and S intertwine with a single left-handed crossover into a coiled-coil at the top, near the globular domains forming a palm tree configuration. This region interacts with the two alpha helices of the second dimer in an opposite orientation to form a four-helix bundle and the dumbbell-shaped tetramer.

Post-translational modifications

In order for hXRCC4 to be sequestered from the cytoplasm to the nucleus to repair a DSB during NHEJ or to complete V(D)J recombination, post-translational modification at lysine 210 with a small ubiquitin-related modifier (SUMO), or sumoylation, is required. SUMO modification of diverse types of DNA repair proteins can be found in topoisomerases, base excision glycosylase TDG, Ku70/80, and BLM helicase. A common conserved motif is typically found to be a target of SUMO modification, ΨKXE (where Ψ is a bulky, hydrophobic amino acid). In the case of the XRCC4 protein, the consensus sequence surrounding lysine 210 is IKQE. Chinese hamster ovary cells, CHO, that express the mutated form of XRCC4 at K210 cannot be modified with SUMO, fail recruitment to the nucleus and instead accumulate in the cytoplasm. Furthermore, these cells are radiation sensitive and do not successfully complete V(D)J recombination.

Interactions

Upon generation of a DSB, Ku proteins will move through the cytoplasm until they find the site of the break and bind to it. Ku recruits XRCC4 and Cer-XLF and both of these proteins interact cooperatively with one another through specific residues to form a nucleoprotein pore complex that wraps around DNA. Cer-XLF is a homodimer that is very similar to XRCC4 in the structure and size of its N-terminal and C-terminal domains. Residues arginine 64, leucine 65, and leucine 115 in Cer-XLF interact with lysines 65 and 99 in XRCC4 within their N-terminal domains. Together they form a filament bundle that wraps around DNA in an alternating pattern. Hyper-phosphorylation of the C-terminal alpha helical domains of XRCC4 by DNA-PKcs facilitates this interaction. XRCC4 dimer binds to a second dimer on an adjacent DNA strand to create a tetramer for DNA bridging early on in NHEJ. Prior to ligation, Lig IV binds to the C-terminal stalk of XRCC4 at the site of the break and displaces the second XRCC4 dimer. The BRCT2 domain of Lig IV hydrogen bonds with XRCC4 at this domain through multiple residues and introduces a kink in the two alpha helical tails. The helix-loop-helix clamp connected to the BRCT-linker also makes extensive contacts.

Mechanism

NHEJ

The process of NHEJ involves XRCC4 and a number of tightly coupled proteins acting in concert to repair the DSB. The system begins with the binding of one heterodimeric protein called Ku70/80 to each end of the DSB to maintain them close together in preparation for ligation and prevent their degradation. Ku70/80 then sequesters one DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the DNA ends to enable the binding of Artemis protein to one end of each DNA-PKcs. One end of the DNA-PKcs joins to stabilize the proximity of the DSB and allow very short regions of DNA complementarity to hybridize. DNA-PKcs then phosphorylates Artemis at a serine/threonine to activate its exonuclease activity and cleave nucleotides at the single strand tails that are not hybridized in a 5' to 3' direction. Two XRCC4 proteins are post-translationally modified for recognition and localization to Ku70/80 (5). The two XRCC4 proteins dimerize together and bind to Ku70/80 at the ends of the DNA strands to promote ligation. XRCC4 then forms a strong complex with DNA ligase IV, LigIV, which is enhanced by Cernunnos XRCC4-like factor, Cer-XLF. Cer-XLF only binds to XRCC4 without direct interaction with LigIV. LigIV then joins the DNA ends by catalyzing a covalent phosphodiester bond.

V(D)J recombination