We have detected DNA polymerase beta (Pol␤), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Pol␤ in the mitochondria. Using Pol␤ fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Pol␤ directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Pol␤ knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Pol␤ mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Pol␤ null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Pol␤ caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Pol␤ is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.KEYWORDS DNA polymerase beta, mitochondrial DNA repair, TFAM, base excision repair, mitochondria, mitochondrial health, mutational studies C ellular DNA repair is critical for genomic stability, and the accumulation of DNA damage has been linked to many debilitating human disorders, including accelerated aging, cancer, and neurodegeneration (reviewed in references 1 and 2). Mammalian cells have two genomes, nuclear and mitochondrial, and both have the ability to replicate, accumulate DNA damage, and propagate mutations. The nucleus contains the vast majority of the mammalian genome and has extensive ability to repair complex bulky adducts, double-strand breaks (DSB), single-strand breaks (SSB), and hundreds of chemical DNA modifications. The ability to effectively repair this breadth of damage is achieved through multiple, often overlapping, DNA repair pathways. In contrast, the repair of mitochondrial DNA (mtDNA) is a more limited version of nuclear DNA (nDNA) repair. Mitochondria lack nucleotide excision repair, and the presence of double-strand break repair is debated (recently reviewed in reference 3). Despite the mitochondria having attenuated DNA repair capabilities compared to the nucleus, the accumulation of mtDNA damage is not without consequence. Ineffective mtDNA maintenance is the underlying cause of many human diseases, including Alpers syndrome and chronic progressive external ophthalmoplegia (CPEO) caused by mutations in mitochondrial polymerase gamma (Pol␥) or the TWINKLE helicase (4-6). The accu-

Patients with CJD232 had no family history like patients with sCJD, and showed two different clinical phenotypes in spite of having the same PRNP genotype. More studies are needed to determine whether M232R substitution causes the disease and influences the disease progression.

SummarySingle-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).

Most cancer cells are aneuploid, which could be caused by defects in chromosome segregation machinery. Nucleoporins (Nup) are components of the nuclear pore complex, which is essential for nuclear transport during interphase, but several nucleoporins are also known to be involved in chromosome segregation. Here we report a novel function of Nup188, one of the nucleoporins regulating chromosome segregation. Nup188 localizes to spindle poles during mitosis, through the C‐terminal region of Nup188. In Nup188‐depleted mitotic cells, chromosomes fail to align to the metaphase plate, which causes mitotic arrest due to the spindle assembly checkpoint. Both the middle and the C‐terminal regions were required for chromosome alignment. Robust K‐fibers, microtubule bundles attaching to kinetochores, were hardly formed in Nup188‐depleted cells. Significantly, we found that Nup188 interacts with NuMA, which plays an instrumental role in focusing microtubules at centrosomes, and NuMA localization to spindle poles is perturbed in Nup188‐depleted cells. These data suggest that Nup188 promotes chromosome alignment through K‐fiber formation and recruitment of NuMA to spindle poles.