Making Ends Meet: This thing called Ku

Ku First discovered as autoantigen in PM/Scl patients Name derived from original patient’s name Antibodies against Ku also found in patients with other autoimmune diseases One of the most abundant DNA end-binding proteins in human cells poly-myositis-scleroderma overlap syndrome

Purified protein binds tightly to free ends of linear dsDNA Recently shown to also bind: ss gaps ss bubbles 5’ or 3’ overhangs hairpin ends When Ku was first isolated by immunoaffinity chromatography using patient antibodies, purified protein was found to tightly bind free ends of linear dsDNA Now known to bind a whole slew of topographic aberrations in dsDNA: ss gaps ss bubbles of noncomplementarity blunt, 5’ overhang, 3’ overhang, hairpin DNA ds ends

Human Ku Heterodimer Conserved across species by size only, Ku70 (69 kDa) Ku80 (83 kDa) Conserved across species by size only, not amino acid sequence Might act as dimer of dimers Ku80 =Ku86 probably arose from duplication and divergence of common ancestor, which might have functioned as homodimer Although two moieties conserved in size across species, aa sequence can differ substantially from one organism to another eg. Mammalian Ku only 20% homology to yeast Ku

Ku70, Ku80, and DNA-PKcs associate to form DNA-PK Two subunits bind tightly to each other, then form a tetramer (or dimer of dimers) when bound to breakage site. After binding, recruits 470 kDa DNA-dependent protein kinase catalytic subunit. 2x Ku70 2xKu80 2xDNA-PKcs = DNA-PK DNA-PK phosphorylates target proteins such as RNA pol II Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

Ku and DNA-PKcs can repair damage caused by physiological oxidative reactions, V(D)J recombination, certain drugs, and ionizing radiation-induced DNA DSBs Ku knock-out mice and yeast reveal additional functions for Ku apart from DNA repair maintenance of genomic integrity Recently (past 5 yrs) shown that DKA-PK play important roles in repair of ionizing radiation-induced DNA DSBs and other damage such as… Multifoliated functions have been attributed to Ku, some of which have dubious functional significance. The best demonstrated functions, from studies in mice and yeast, involve Ku in maintenance of genomic integrity.

NHEJ proteins in S. cerevisiae and human cells Is this slide needed?

Linking Ku with DNA DSB repair In mammalian systems 1994 - DNA-PKcs- & Ku80-deficient cells have defective DNA DSB rejoining extreme sensitivity to ionizing radiation and other agents that cause DNA DSBs less sensitive to UV, alkylating agents, mitomycin C Ku70 knock-out phenotype hypersensitive to ionizing radiation defective DNA-end binding activity due to Ku cannot support V(D)J recombination Until about 5 yrs ago, Ku was studied primarily as modulator of transcription because it phosphorylates transcription factors such as Sp1, p53, Oct-1 But focus was shifted after the discovery that two complementation groups of x-ray-sensitive mammalian cell lines are deficient in DNA-PK activity and can be complemented by genes encoding Ku80 (XRCC5 gene) and DNA-PKcs (XRCC7 gene). These cell lines have defective DNA DSB rejoining and are sensitive to ionizing radiation and other DSB-causing factors. Less sensitive to other types of DNA damage from UV, alkylating agents, mito C. Ku70 disruption causes hypersensitivity to ionizing radiation… Together, these findings demonstrate Ku plays a vital role in DNA DSB repair.

SCID (severe combined immuno-deficiency) mice radiosensitive, defective in DSB repair characteristic of a DNA-PKcs defect radiosensitivity complemented by XRCC7 (DNA-PKcs) gene immunodeficiency due to V(D)J defect cells cannot properly rearrange immunoglobulin and T-cell receptor gene segments cannot maturate and diversify antibodies and T-cell receptors Ku70 or Ku80 knock-outs have immuno-deficiency phenotype similar to SCID Around this time, SCID studies showed radiosensitivity and defect in DSB repair phenotype is due to mutation in DNA-PKcs gene (XRCC7). (2nd point) SCID mice also are immunodeficient due to V(D)J defect. V(D)J requires formation and rejoining of DNA DSBs, and rejoining step is dysfunctional in SCID. Variable, diversity, joining regions of immunoglobulin and t-cell receptor genes cannot properly rearrange to give rise to mature, functional antigen receptors on surface of precursor B- and T- cells. They arrest early in development and therefore SCID mice lack mature functional B, T cells. Complementary to these findings, mice engineered to be deficient in Ku70 or Ku80 have an immunodeficiency phenotype similar to SCID.

All components of DNA-PK function in generating diverse antigen-binding functions of mammalian immune system Thus...

In cerevisiae Heterodimer functions in NHEJ ligates two DNA ends without extensive homology little or no nucleotide loss Although NHEJ repairs most vertebrate DSBs, in yeast repaired mainly by homologous recombination NHEJ important in haploid G1 no homologous chromosomes present for homologous recombination In Saccharomyces cerevisiae, homoloues of Ku70 (yKu70p or Hdf1p) and Ku80 (yKu80p or Hdf2p) also form heterodimer involved in DNA DSB repair. This pathway is NHEJ or illegitimate recombination, which is very different from homologous recombination because… Point 2. This might seem counterintuitive at first, but Point 3. Also, there are indications that yeast Ku repairs restriction enzyme-induced chromosomal damage.

Impair yKu70p or yKu80p, severely impair NHEJ But no obvious DNA-PKcs homologue functions mediated by DNA-PKcs do not occur in yeast mediated by other polypeptides Mec1p, Tel1p Point 1. The joining reactions that do occur (at low levels) is error prone and produces deletions of up to several hundred bp’s. Similar types of repair products can be seen in the low level of V(D)J recombination that occurs in absence of Ku in mammalian cells, suggestive of a conservation of Ku system and its imperfect backup pathway. But there is really no DNA-PKcs homologue in cerevisiae… (Mec1p, Tel1p are DNA-PKcs related proteins)

How does Ku function in DNA DSB repair? Ku binds tightly and rapidly to DNA ends likely Ku can recognize various broken DNA structures in cells might prevent exonuclease activity on DNA but V(D)J intermediates stable without Ku possibility: Ku holds two DNA ends on both sides of DSB facilitates processing and ligation by other repair components Since… Ku binds tightly and rapidly to DNA ends, it is likely that Ku can recognize various broken DNA structures in cells. Once bound, Ku might act to prevent exonuclease digestion on the ends. But V(D)J intermediates are relatively stable in the absence of Ku. Because Ku can transiently bridge two DNA molecules...

Can Ku function in targeting nucleases (Rad50p, Mre11p) to DSB site and/or modulate nuclease activities? SbcC, SbcD act as nucleases in E. Coli RAD50, MRE11, XRS2 form epistasis group required for NHEJ in yeast Usually, though, DSBs cannot simply be ligated together but require pre-processing. So… Rad50p = SbcC Mre11p = SbcD SbcC & SbcD act together in E. Coli as a nuclease Also, RAD50, MRE11, and XRS2 genes form a single epistasis group that’s required for NHEJ in yeast. There are mammalian homologues for Rad50p and Mre11p that have nuclease activities in vitro, so these proteins appear to be evolutionarily conserved. In mammals, Rad50p homologue, Mre11p homologue, and a third factor called NBS1 form a complex which, when disrupted, presents with radiosensitivity, developmental abnormalities, and cancer predisposition.

Ku can translocate along DNA in ATP-independent fashion each dimer binds to DNA end slides apart from each other to open helix Ku has weakly processive DNA helicase activity ends presented with regions of microhomology ends anneal together Another property reported for Ku is that it (Point 1). Each dimer, when bound to ends of DNA, may slide apart, locally dissociating two strands, allowing for regions of microhomogy at ends to anneal together. I should mention that Ku has weakly processive DNA helicase activity, which acts to aid in the local melting of DNA ends. This microhomology-mediated annealing might be followed by exonuclease attack on unpaired tails by Rad50-Mre11 complex, gap filling by DNA pol, and ligation by DNA ligase IV.

DNA-PK in NHEJ To repeat, Ku binds, DNA-PKcs tethers DNA ends while Ku melts DNA ends, strands anneal due to microhomology, Rad50-Mre11 nuclease chews up ss ends left over, DNA pol and DNA ligase IV fill in the gaps. Once DNA ends are ligated, , DNA-PK autophosphorylates and dissociate from the DNA. This model is nice because it satisfactorily explains small deletions often observed at sites of NHEJ. Also, DNA-PKcs probably recruits other DSB repair components to site of damage and/or regulate them by phosphorylation. Kinase activity of DNA-PK could also signal presence of DNA damage, resulting in cell cycle arrest or even initiating apoptosis cascade. DNA-PK has been shown to be involved in inducing p53 DNA binding activity after exposure to DNA damaging agents. Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.

Ku is implicated in transcription DNA-PK phosphorylates transcription factors and regulatory C-terminal domain of RNA polymerase II in vitro no evidence yet that transcriptional proteins act as substrates for Ku in vivo Ku binds sequences in transcriptional regulatory elements no clear consensus sequence for Ku DNA-binding Ku has also been implicated in transcription. Several lines of experimental evidence have arisen...

DNA-PK can phosphorylate RNA polymerase I transcription apparatus responsible for transcription of large ribosomal RNA precursor Ku binding changes local conformation of DNA substrate equilibrium shifts from euchromatin to heterochromatin might repress transcription might facilitate juxtaposition of DNA ends Point 1, point 1b. This only happens in the presence of DSBs, so it might attenuate transcription of ribosomal genes if DSB occurs within these loci. Point 2. In yeast, it has been shown that the silencing proteins Sir2p, Sir3p and Sir4p are required for NHEJ. Furthermore, it has been shown by two-hybrid assay that yKu70p interacts with Sir4p. Ku recruits Sir proteins to DSBs, Sir proteins generate condensed, heterochromatin-like state around DSB. Such a conformation would repress transcription as well as other processes that could interferewith DSB repair. Chromatin condensation could also facilitate the juxtaposition and subsequent ligation of the two DNA ends.

Physiological functions of Ku Ku70, Ku80 knockouts in mice have similar phenotype to SCID V(D)J defects arrest lymphocyte development Ku70, Ku80 -/- mice are runts compared to +/- littermates Number of cell divisions in development limited by impaired ability to repair endogenously generated DNA damage Ku-deficient cells might take longer to repair this damage Ku80 -/- dams fail to nurture their pups We already know that (Point 1). Point 2. 40-60% size of littermate controls…. Ku80 -/- females are unable to sustain their pups, which die within a few days unless they are nursed by a foster mother. This finding was from a Letter to Nature, and the authors did not expound on why this is so.

Yeast Ku in telomere maintenance Disruption of yKu70p and yKu80p genes affect telomeric silencing and telomere length maintenance inactivate Ku, lose telomeric silencing inactivate Ku, shorten telomeres Model: Ku binds double-stranded telomeric ends, blocks accessibility of certain nucleases in most of cell cycle. Ku displaced from telomeric ends during S phase, allowing exonucleolytic degradation of one strand, creating ssDNA binding site for telomerase When the genes that encode either Yku70p or yKu80p are disrupted, not only NHEJ but also telomeric silencing and telomere length maintenance are significantly perturbed. Telomeric silencing (aka telomere position effect) occurs when a gene is engineered into the telomeric region of a yeast chromosome. In these end regions, the chromatin is in a unique condensed structure that doesn’t permit access by the transcriptional apparatus and therefore the engineered gene is silent. If this heterochromatin-like state is disrupted, telomeric silencing is relieved and genes can be expressed. When Ku is inactivated, this occurs as well as the loss of about 65% of telomere length. How is this possible? Perhaps Ku protects telomeric DNA ends from degradation. Point 2.

Ku clusters yeast telomeres to peripheral sites in nucleus In diploids, telomeres usually found in 6-7 clusters around nuclear periphery In Ku subunit mutants, more clusters in random locations At another level, Ku appears to be involved in the clustering of yeast telomeres at peripheral sites in the nucleus. In wild-type diploid yeast cells, the 63 telomeres are usually found in six or seven clusters around the nuclear periphery, while cells mutant in either Ku subunit gene have around nine clusters that seem to be located more randomly throughout the nucleus. These results suggest Ku is somehow involved in clustering telomeres and tethering them at sites in the nuclear periphery. Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.