Artemis is required to improve the accuracy of repair of double-strand breaks with 5'-blocked termini generated from non-DSB-clustered lesions.

Clustered DNA lesions are defined as ≥2 damage events within 20 bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART(-/-)) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5' termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART(-/-) cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5'-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.

DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining.

Clustered lesions are defined as >or=two lesions within 20 bps and are generated in DNA by ionizing radiation. In vitro studies and work in bacteria have shown that attempted repair of two closely opposed lesions can result in the formation of double strand breaks (DSBs). Since mammalian cells can repair DSBs by non-homologous end-joining (NHEJ), we hypothesized that NHEJ would repair DSBs formed during the removal of clustered tetrahydrofurans (furans). However, two opposing furans situated 2, 5 or 12 bps apart in a firefly luciferase reporter plasmid caused a decrease in luciferase activity in wild-type, Ku80 or DNA-PKcs-deficient cells, indicating the generation of DSBs. Loss of luciferase activity was maximal at 5 bps apart and studies using siRNA implicate the major AP endonuclease in the initial cleavage. Since NHEJ-deficient cells had equivalent luciferase activity to their isogenic wild-type cells, NHEJ was not involved in accurate repair of clustered lesions. However, quantitation and examination of re-isolated DNA showed that damage-containing plasmids were inaccurately repaired by Ku80-dependent, as well as Ku80-independent mechanisms. This work indicates that not even NHEJ can completely prevent the conversion of clustered lesions to potentially lethal DSBs, so demonstrating the biological relevance of ionizing radiation-induced clustered damage.

Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology.

Unlike Escherichia coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of microhomology (1-4bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.

DNA repair of clustered uracils in HeLa cells.

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.

Two clustered 8-oxo-7,8-dihydroguanine (8-oxodG) lesions increase the point mutation frequency of 8-oxodG, but do not result in double strand breaks or deletions in Escherichia coli.

Multiply damaged sites (MDSs) are generated in DNA by ionizing radiation. In vitro studies predict that base excision repair in cells will convert MDSs to lethal double strand breaks (DSBs) when two opposing base damages are situated >/=2 bp apart. If the lesions are situated immediately 5' or 3' to each other, repair is predicted to occur sequentially due to inhibition of the DNA glycosylase by a single strand break repair intermediate. In this study, we examined how the distance between two opposing lesions alters the mutation frequency of an 8-oxodG in an MDS, and whether repair generates DSBs and deletions in bacteria. The 8-oxodG mutation frequency declined in MutY-deficient bacteria when the opposing 8-oxodG was 6 bp away, and was similar to a single 8-oxodG when the lesions were separated by 14 bp. However, the number of deletions detected for the MDSs was equivalent to the undamaged sequence. Using a separate assay, MDSs consisting of two 8-oxodG or an 8-oxodG opposite a uracil were not converted to DSBs in the absence of DNA replication in wild-type and transcription-coupled repair-deficient bacteria. This is the first study showing that DSB-repair intermediates and deletions are not formed during repair of clustered 8-oxodGs in cells.

The mutation frequency of 8-oxo-7,8-dihydroguanine (8-oxodG) situated in a multiply damaged site: comparison of a single and two closely opposed 8-oxodG in Escherichia coli.

A multiply damaged site (MDS) is defined as > or =2 lesions within a distance of 10-15 base pairs (bp). MDS generated by ionizing radiation contain oxidative base damage, and in vitro studies have indicated that if the base damage is <3bp apart, repair of one lesion is inhibited until repair of the lesion in the opposite strand is completed. Inhibition of repair could result in an increase in the mutation frequency of the base damage. We have designed an assay to determine whether a closely opposed lesion causes an increase in adenine insertion opposite an 8-oxodG in bacteria. We have positioned the MDS (an 8-oxodG in the transcribed strand and a second 8-oxodG immediately 5' to this lesion in the non-transcribed strand) within the firefly luciferase coding region. During two rounds of replication, insertion of adenine opposite the 8-oxodG in the transcribed (T) or non-transcribed (NT) strand results in a translation termination codon at position 444 or 445, respectively. The truncated luciferase protein is inactive. We have generated double-stranded oligonucleotides that contain no damage, each single 8-oxodG or the MDS. Each double-stranded molecule was ligated into the reporter vector and the ligation products transformed into wild-type or Mut Y-deficient bacteria. The plasmid DNA was isolated and sequenced from colonies that did not express luciferase activity. In wild-type bacteria, we detected a translation stop at a frequency of 0.15% (codon 444) and 0.09% (codon 445) with a single 8-oxodG in the T or NT strand, respectively. This was enhanced approximately 3-fold when single lesions were replicated in Mut Y-deficient bacteria. Positioning an 8-oxodG in the T strand within the MDS enhanced the mutation frequency by approximately 2-fold in wild-type bacteria and 8-fold in Mut Y-deficient bacteria, while the mutation frequency of the 8-oxodG in the NT strand increased by 6-fold in Mut Y-deficient bacteria. This enhancement of mutation frequency supports the in vitro MDS studies, which demonstrated the inability of base excision repair to completely repair closely opposed lesions.

A novel MeCP2 acetylation site regulates interaction with ATRX and HDAC1.

Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used to selectively decrease H3K27me3 repressive marks on a subset of repressed target gene promoters analyzed. Lastly, we show that RKO cells over-expressing MeCP2 mutant show reduced proliferation compared to those over-expressing MeCP2-wildtype. Our study demonstrates the importance of acetylated lysine residues and suggests their key role in regulating MeCP2 function and its ability to bind transcriptional regulators.

Can DNA repair cause enhanced cell killing following treatment with ionizing radiation?

Production of DNA damage is the basis of cancer treatments, such as chemotherapy and radiotherapy. The limitation of the treatment dose tends to be how well the normal cells within the body can tolerate the therapy. Although it is possible, to some extent, to localize the treatment area during radiotherapy by targeting the beam of ionizing radiation, chemotherapy usually involves a whole body treatment. In order to improve the effectiveness of treatments, it is important to understand how cells repair the DNA damage. This review will attempt to explain how DNA repair, which would be expected to always enhance cell survival, actually may result in increased cell killing following certain types of cancer treatments, such as ionizing radiation and bleomycin sulfate. Work is underway in many laboratories to unravel how the repair systems handle specific types of DNA damage. Such information will pave the way in designing adjuvant therapies that alter a tumor cell's DNA repair capacity and increase tumor cell killing.

SIRT1 positively regulates breast cancer associated human aromatase (CYP19A1) expression.

Breast cancer remains one of the leading causes of death in women diagnosed with cancer. In breast cancer, aberrant expression of the CYP19A1 gene, which encodes the aromatase enzyme, contributes to increased intratumoral levels of estradiol. Regardless of whether this estrogen is produced by peripheral tissues or within specific subpopulations of cells within the breast tumor, it is clear that the aromatase enzymatic activity is critical for the growth of estrogen-dependent tumors. Currently, aromatase inhibitors have proven to be highly effective in blocking the growth of estrogen-dependent forms of breast cancer. CYP19A1 transcription is tightly controlled by 10 tissue-specific promoters. In breast cancer, however, aromatase transcription is driven by multiple promoters that somehow override the tissue-specific regulation of normal tissue. Here, we explore the role that the deacetylase, sirtuin-1 (SIRT1), plays in positively regulating aromatase in breast cancer. We demonstrate that the use of cambinol and the SIRT1/2 inhibitor VII, 2 small molecule inhibitors of SIRT1 and SIRT2, as well as small molecule inhibitors and small interfering RNA specific to SIRT1, all reduce the levels of aromatase mRNA. We further demonstrate that pharmacologic inhibition causes a marked reduction in aromatase protein levels. Additionally, by chromatin immunoprecipitation, we demonstrate that SIRT1 occupies the promoter regions PI.3/PII and PI.4, and its inhibition leads to increased acetylation of estrogen-related receptorα, a transcription factor that positively regulates CYP19A1 transcription in epithelial cells. Finally, we demonstrate by immunohistochemistry that SIRT1 is significantly up-regulated in invasive ductal carcinoma relative to normal tissue adjacent to tumor, further suggesting a role of SIRT1 in breast cancer. This work uncovers a new mechanism for the regulation of aromatase and provides rationale for further investigation of how the inhibition of specific sirtuins may provide a unique strategy for inhibiting aromatase that may complement or synergize with existing therapies.

Apex1 can cleave complex clustered DNA lesions in cells.

Current data indicate that clustered DNA damage generated by ionizing radiation contains 2-5 damages within 20 bps. The complexity of clustered damage is also believed to increase as the linear energy transfer of the radiation increases. Complex lesions are therefore biologically relevant especially with the use of carbon ion beam therapy to treat cancer. Since two closely opposed AP site analogs (furans) are converted to a double strand break (DSB) in cells, we hypothesized that breakage could be compromised by increasing the complexity of the cluster. We have examined the repair of clusters containing three and four lesions in mouse fibroblasts using a luciferase reporter plasmid. The addition of a third furan did reduce but not eliminate cleavage, while a tandem 8-oxo-7,8-dihydroguanine (8oxoG) immediately 5' to one furan in a two or three furan cluster decreased DSB formation by a small amount. In vitro studies using nuclear extracts demonstrated that the tandem 8oxoG was not removed under conditions where the furan was cleaved, but the presence of the 8oxoG reduced cleavage at the furan. Interestingly, a cluster of an 8oxoG opposite a furan did not form a DSB in cells. We have shown that Apex1 can cleave these complex clustered lesions in cells. This therefore indicates that Apex1 can generate complex DSBs from clustered lesions consisting of base damage and AP sites. Repair of these complex DSBs may be compromised by the nearby oxidative damage resulting in potentially lethal and biologically relevant damage.