The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.

Repair of DNA double-strand breaks in mammalian cells occurs via a direct nonhomologous end-joining pathway. Although this pathway can be studied in vivo and in crude cell-free systems, a deeper understanding of the mechanism requires reconstitution with purified enzymes. We have expressed and purified a complex of two proteins that are critical for double-strand break repair, DNA ligase IV (DNL IV) and XRCC4. The complex is homogeneous, with a molecular mass of about 300,000 Da, suggestive of a mixed tetramer containing two copies of each polypeptide. The presence of multiple copies of DNL IV was confirmed in an experiment where different epitope-tagged forms of DNL IV were recovered simultaneously in the same complex. Cross-linking suggests that an XRCC4⅐XRCC4 dimer interface forms the core of the tetramer, and that the DNL IV polypeptides are in contact with XRCC4 but not with one another. Purified DNL IV⅐XRCC4 complex functioned synergistically with Ku protein, the DNA-dependent protein kinase catalytic subunit, and other repair factors in a cell-free end-joining assay. We suggest that a dyad-symmetric DNL IV⅐XRCC4 tetramer bridges the two ends of the broken DNA and catalyzes the coordinate ligation of the two DNA strands.

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70-and 86-kDa heterodimeric regulatory component, also known as the Ku protein. We report here that HSF1 binds specifically to each of the two components of DNA-PK. Binding occurs in the absence of DNA. The complex with the Ku protein is stable and forms at a stoichiometry close to unity between the Ku protein heterodimer and the active HSF1 trimer. The binding is blocked by antibodies against HSF1. Our results show that HSF1 also binds directly, but more weakly, to the catalytic subunit of DNA-PK. Both interactions are dependent on a specific region within the HSF1 regulatory domain. This sequence is necessary but not sufficient for HSF1 stimulation of DNA-PK activity. The ability of HSF1 to interact with both components of DNA-PK provides a potential mechanism for the activation of DNA-PK in response to heat and other forms of stress.

Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle. Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair protein, and that activity is rescued by expression of the corresponding cDNA. We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA. The factor may consist of a preformed complex of transcription proteins that is stabilized by Ku. A Ku mutant, lacking residues 687-728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with proteins required for reinitiation. The Ku mutant functions normally in a DNA endjoining system, indicating that the functions of Ku in transcription and repair are genetically separable. Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.The ability of a cell to produce multiple copies of a particular mRNA requires the recycling of template, general transcription factors, and RNAP II. 1 This process, termed reinitiation, is a key determinant of the overall transcription rate. Reinitiation at a given promoter occurs in vivo as frequently as every few seconds (1, 2). The ability to maintain such high, sustained transcription rates suggests the existence of a facilitated reinitiation pathway in which many sequential events are coordinated so that the transcription cycle can proceed at a rapid overall rate (reviewed in Ref. 3).Mechanisms that promote rapid reinitiation can be divided broadly into those that operate at the template level, influencing the availability of a particular DNA for reinitiation, and those that operate at the protein level, influencing the availability of RNAP II and transcription factors. A number of examples of mechanisms that promote reinitiation have been characterized. One of the mechanisms that operates at the template level is the persistent binding of transcription factors to the promoter. The binding of TFIID and TFIIA is commonly the rate-limiting step in de novo transcription complex formation (4 -6). After the first round of initiation is complete, TFIID and TFIIA remain bound to the DNA and nucleate the assembly of another transcription complex (7). Because this bypasses a potentially rate-limiting step, the overall rate of reinitiation is increased. Consistent with this interpretation, mutations that destabilize the TFIID-TFIIA-DNA complex selectively decrease the reinitiation rate in vitro (8, 9). Another example of a mechanism that promotes the availability of templates for reinitiation is the suppression of pausing during elongation. The presence of a stable, paused elongation...

In human peripheral interstitial fluid, esterification of cholesterol by lecithin cholesterol acyltransferase (LCAT) was found to occur at a rate of only 10% of that in plasma (5.6±1.8 compared with 55.6±7.8 nmol/ml per h). Measurement of cholesterol esterification in the presence of excess reconstituted apoA-I HDL (rA-I HDL) revealed an LCAT activity in interstitial fluid of 24% of that in plasma, indicating that the low rate of esterification could not be caused by limiting mass of LCAT enzyme. When plasma was diluted to the same concentration as in interstitial fluid, the percent cholesterol esterification rate was the same as undiluted plasma and significantly higher than that of interstitial fluid. These findings led us to postulate that poor activation of LCAT in interstitial fluid may result from a change in conformation in apoA-I. To test this hypothesis, a monoclonal antibody Al-11 that inhibits apoA

We studied the Interstitial fluid concentration of two llpld-metabollzing enzymes (lipoprotein lipase and hepatic triacylglycerol lipase) to determine their Importance in Interstitial modification of filtered llpoprotelns. Despite the use of a very sensitive lipase assay (1 nmol of fatty acid release/ml/hr), lipase activities in plasma and In peripheral and skeletal muscle lymph from control dogs were below the sensitivity of our assay. After heparin Injection, hepatic triacylglycerol lipase and lipoprotein lipase activities In plasma were similar. However, the postheparin hepatic triacylglycerol lipase activities in peripheral and skeletal muscle lymph were only 1.4% and 1.1%, respectively, those of plasma. This concentration Is considerably less than the lymph concentration of albumin, which has a similar size to the Upases but has a lymph concentration of 30% to 40% of plasma. Lipoprotein lipase activity In peripheral lymph and skeletal muscle lymph was 2.7% and 4.8%, respectively, of plasma activity. Since lipoprotein lipase has a similar size as hepatic triacylglycerol lipase, the disproportionate amount of lipoprotein lipase In lymph as compared to hepatic triacylglycerol lipase could be due to heparin crossing the capillary ondothollum and displacing lipoprotein lipase from peripheral cells. Injection of radioactive heparin confirmed that It does cross Into the interstitial space In sufficient concentrations to displace lipase from peripheral cells. We conclude that most of the lipase found In lymph after heparin Injection Is derived from peripheral cells and not from plasma. Furthermore, hepatic triacylglycerol lipase does not play a role In high density lipoprotein remodeling In interstitial fluid. Therefore, it seems likely that the considerable remodeling of high density lipoprotein that we found previously results from its Interaction with peripheral cells.

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