Leukemia inhibitory factor (LIF) has been recently identified as a p53 target gene, which mediates the role of p53 in maternal implantation under normal physiological conditions. Here, we report that LIF is a negative regulator of p53; LIF downregulates p53 protein levels and function in human colorectal cancer (CRC) cells. The downregulation of p53 by LIF is mediated by the activation of Stat3, which transcriptionally induces ID1. ID1 upregulates MDM2, a key negative regulator of p53, and promotes p53 protein degradation. LIF is overexpressed in a large percentage of CRCs. LIF overexpression promotes cellular resistance towards chemotherapeutic agents in cultured CRC cells and colorectal xenograft tumors in a largely p53-dependent manner. Overexpression of LIF is associated with a poor prognosis in CRC patients. Taken together, LIF is a novel negative regulator of p53, overexpression of LIF is an important mechanism for the attenuation of p53, which promotes chemoresistance in CRCs.

On the assumption that Rad51 protein plays a role in early meiotic chromosomal events, we examine the location and time of appearance of immuno-reactive Rad51 protein in meiotic prophase chromosomes. The Rad51 foci in mouse spermatocytes appear after the emergence of, and attached to, short chromosomal core segments that we visualize with Cor1-specific antibody. These foci increase in number to about 250 per nucleus at the time when core formation is extensive. The numbers are higher in mouse oocytes and lower in rat spermatocytes, possibly correlating with recombination rates in those cases. In the male mouse, foci decrease in number to approximately 100 while chromosome synapsis is in progress. When synapsis is completed, the numbers of autosomal foci decline to near 0 while the X chromosome retains about 15 foci throughout this time. This stage coincides with the appearance of testis-specific histone H1t at mid- to late pachytene. Electron microscopy reveals that at first Rad51 immunogold-labeled 100 nm nodules are associated with single cores, and that they come to lie between the chromosome cores during synapsis. It appears that these nodules may be the homologs of the Rad51-positive early nodules that are well documented in plants. The reciprocal recombination-correlated late nodules appear after the Rad51 foci are no longer detectable. The absence of Rad51 foci in the chromatin loops suggests that in wild-type mice Rad51/DNA filaments are restricted to DNA at the cores/synaptonemal complexes. The expected association of Rad51 protein with Rad52 could not be verified immunocytologically.

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.NA double strand breaks (DSBs) are a most severe type of DNA damage caused by endogenous metabolic processes and exogenous exposure to radiation and chemicals. Unrepaired DSBs induce genomic instability, carcinogenesis, and premature aging. In mammalian cells, DSBs are repaired by either the nonhomologous end joining (NHEJ) or the homologous recombination (HR) pathway. Although it is a common understanding that HR primarily takes place in response to strand breaks in the S-G2 phases of the cell cycle where the undamaged sister chromatids are present as donor templates, recent studies have suggested that homologous pairing also occurs during the G0/G1 phase and is associated with transcription (1), although the mechanisms remain to be elucidated. At active transcription sites, RNA polymerase II (RNA POLII) can bypass base modifications such as 8-oxo guanine but not single strand breaks (SSBs) and DSBs (2-5), indicating that unrepaired strand breaks at transcriptionally active (TA) sites can be especially deleterious and may lead to secondary damage.The Cockayne syndrome B (CSB) gene is defective in approximately two-thirds of patients with Cockayne syndrome (CS), an autosomal recessive disease with diverse clinical signs including severe growth failure, progressive neurodegeneration, and hypersensitivity to sunlight. CSB has an established role in transcription-coupled nucleotide excision repair (TC-NER) of photo lesions. When RNA POLII is stalled at bulky lesions, CSB is loaded to facilitate NER of the transcribed strand (6, 7). As noted, in addition to UV sensitivity, CS patients also manifest severe neurodegeneration (8, 9), suggesting the importance of CS proteins in maintaining genome stability against a broad spectrum of DNA damage. For example, CSB-defective cells are also sensitive to ionizing radiation (IR) (10, 11), which is phenotypically distinctive from classic NER deficiencies and indicates that CSB function is not limited to UV-derived photo lesions. In addition, CSB-deficient mice exhibit a subset of sy...

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Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIP␣ interacts with BRCA2. We show that a second isoform, BCCIP␤, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIP␣ and BCCIP␤. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIP␣ by 90% or BCCIP␤ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20-to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2-and RAD51-dependent responses to DNA damage and HRR.DNA double-strand breaks (DSBs) are induced by exogenous agents, such as ionizing radiation (IR), and arise spontaneously during normal DNA metabolism, such as at blocked or collapsed replication forks (9,10,39,45). Defects in DSB repair confer genome instability associated with tumorigenesis. In mammalian cells, DSBs are repaired by nonhomologous end-joining and by homologous recombinational repair (HRR) (60,62,65). RAD51 binds single-stranded DNA (ssDNA) to form nucleoprotein filaments that are essential for strand transfer during HRR (23,44,61,66). RAD51 is normally dispersed in the nucleus, but upon DNA damage induction, it redistributes to nuclear foci that are presumed sites of HRR (6,7,14,20,31,46). RAD51 foci have been shown to be associated with ssDNA regions after DNA damage (46). Several HRR proteins, including XRCC2, XRCC3, RAD51B, RAD51C, RAD51D, and BRCA2, are important for RAD51 focus formation (1,5,7,43,55,56).BRCA2 has nine RAD51 binding regions, including eight BRC repeats encoded by exon 11 and a distinct RAD51 binding region encoded by exon 27 (8, 33, 69). Expression of individual BRC repeats interferes with RAD51 focus formation and HRR (5, 53, 70), indicating that RAD51-BRCA2 interactions are important for both processes. The C-terminal half of BRCA2 has three regions that are structurally related to the ssDNA binding region of RPA and bind ssDNA in vitro, suggesting that ssDNA binding is also important for BRCA2 function in HRR (71). These ssDNA binding regions occur in a region called conserved domain IV (30,48,73) or the BRCA2 C-terminal domain (71), which is the longest and most evolutionarily conserved BRCA2 domain (32, 57). This domain also has binding sites for several proteins including DSS1, BUBR1, ABP-280/filamin-A, and BCCIP␣ (16,30,34,73).BCCIP␣ is a BRCA2 and CDKN1A (p21, Cip1, and Waf1) interaction protein (30); it has also been called . A second isoform, BCCIP␤, shares an N-terminal acidic domain and a central conserved domain but has a distinct C-terminal domain (Fig. 1A). In this report, BCCIP indicates both proteins. The BCCIP proteins share no significan...

Genomic instability refers to an increased tendency of alterations in the genome during the life cycle of cells. It is a major driving force for tumorigenesis. During a cell division, genomic instability is minimized by four major mechanisms: high-fidelity DNA replication in S-phase, precise chromosome segregation in mitosis, error free repair of sporadic DNA damage, and a coordinated cell cycle progression. This introduction summarizes the major molecular processes that contribute to these mechanisms in the context of prevention of genomic instability and tumorigenesis.

Processing of DNA damage by the DNA double-strand break repair pathway in mammalian cells is accomplished by multiprotein complexes. However, the nature of these complexes and details of the molecular interactions are not fully understood. Interaction of the yeast RAD51 and RAD52 proteins plays a crucial role in yeast DNA homologous recombination and DNA double-strand break repair. Here, specific interactions between human RAD51 and RAD52 proteins are demonstrated both in vivo, using the yeast two-hybrid system and immunoprecipitation of insect cells co-infected with RAD51 and RAD52 recombinant viruses, and in vitro, using affinity chromatography with purified recombinant proteins. These results suggest that RAD52 may modulate the catalytic activities of RAD51 protein such as homologous pairing and strand exchange through a direct physical interaction. In addition, the domain in RAD52 that mediates this interaction was determined in vitro and in vivo. The RAD51-interacting region (amino acids 291-330) of the human RAD52 protein shows no homology with the yeast RAD52 protein, indicating that the interaction between RAD51 and RAD52 is species-specific.

The RAD51/RAD52-dependent DNA repair pathway is involved in DNA recombination and DNA double-strand break repair in yeast. Although many proteins in the RAD51/RAD52-dependent DNA repair pathway have been identified in yeast, a novel protein(s) that functions with RAD51/RAD52 may also exist in humans. Using a yeast two-hybrid system, we have identified a 12-kDa protein that associates with the human RAD51 and RAD52 proteins. This protein shares significant amino acid homology with the yeast protein SMT3, which functionally associates with the yeast mitosis fidelity protein MIF2. It also shares moderate homology with ubiquitin and several other proteins, including the N-terminus of the RAD23 protein and a ubiquitin cross reacting protein. Therefore, the gene is tentatively designated UBL1 for ubiquitin-like 1. The UBL1 mRNA is expressed in many human tissues, most highly in testis. The UBL1 gene is mapped to chromosome 2q32.2-q33, and a related sequence may be located on chromosome 1q23-q25.