In contrast, CHST3 knock-out mice and have neither skeletal nor cardiac defects

ould inhibit AP site repair in wild type cells or animals. One possibility is that one subunit is degraded while leaving the other intact. Granzyme A cleaves Ku70 at Arg301 to separate the two Ku80 binding domains thereby preventing formation of the Ku heterodimer as a part of the caspaseindependent cell death pathway in killer cell cytotoxic granules. Interestingly, GzmA also cleaves APE1 to induce cell death; thus, Ku70 cleavage could further diminish BER. Since GzmA cleaves Ku70 at Arg301, we tested myc-Ku701300 for its binding preference to these substrates. Similar to full length Ku70, Myc-Ku701300 exhibited preferential binding to the AP/G substrate as compared to the C/G and U/G substrates. In addition, a competition assay with myc-Ku701300 was performed with hot and cold substrate. We found 30X cold AP/G competed out hot AP/G when compared to no competitor. However, 5 Deletion of Ku Interferes with AP Site Repair 100X cold C/G and 100X cold U/G did not compete with the AP substrate to the same extent as 30X cold AP/G. Thus, Ku701300 preferentially bound to AP sites. We also found that preferential binding to the AP/G substrate was seen for myc-Ku701115 as compared to the C/G and U/G substrates. However, myc-Ku70115609 did not display preferential binding to the AP/G as compared to the C/G and U/G substrates. Thus, myc-Ku701115 was necessary and sufficient to preferentially bind to the AP/G substrate, suggesting the N-terminal Ku70 cleavage product associates with AP sites to inhibit APE1. Full length and N-terminal Ku70, but not C-terminal Ku70 or Ku80, inhibited APE1 activity Free Ku70 and free Ku80 binding to AP sites suggest a possible mechanism for interfering with APE1 activity. This activity was tested using a real-time molecular beacon assay that measures fluorescence emitted after purified APE1 nicks 59 to the AP site to release a FAM fluorophore from the dabsyl quench located on the complementary DNA strand. APE1 effectively released the FAM fluorophore from the DNA substrate without Ku70. Addition of myc-Ku701609 prohibited APE1 nicking. However, addition of myc-Ku70115609 did not affect APE1 nicking. Furthermore, addition of myc-Ku701115 myc-Ku701300 impaired APE1 as compared to no myc-Ku70, though not as efficiently as full-length myc-Ku70. These data indicate that myc-Ku701115, mycKu701300 and myc-Ku701609, but not myc-Ku70115609, inhibited APE1 activity in keeping with the AP binding data. Thus, Ku70 binding to AP sites inhibits APE1 nicking. Interestingly, Ku80 did not inhibit APE1 activity by this assay, though a previous report showed that Ku and DNA-PKCS inhibited AP site cleavage by APE1. Disscussion Mice and cells deleted for either Ku70 or Ku80 exhibit a complex phenotype that is not solely due to disruption of NHEJ; it is possible some of this complexity is due to altered BER. In mice Ku80-deletion had a more severe order SB-203580 impact on life span than DNAPKCS-deletion suggesting Ku80 does more PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19649022 than NHEJ. The level of small mutations also changed in mouse brains deleted for either Ku70 or Ku80 implicating a change in BER. To further implicate altered BER, cells deleted for either Ku70 or Ku80, but not Lig4, exhibited increased sensitivity to ROS and alkylating agents and cells deleted for Ku80, but not Lig4, were deficient in repairing AP sites and cell extracts were deficient in correcting a U/G mismatch in an oligonucleotide substrate. Furthermore, ectopic expression of either OGG1 or PARP-1 recused hypersensitivity