Kristine E. Yoder

DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics

ABSTRACT Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.

Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection

Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell. We report that unintegrated viral cDNA is a substrate for the host cell non‐homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double‐strand breaks by end ligation. NHEJ activity was found to be required for an end‐ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells. Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates. Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration. We propose that the double‐strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double‐strand breaks, and cDNA circularization removes the pro‐apoptotic signal.

Repair of Gaps in Retroviral DNA Integration Intermediates

ABSTRACT Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.

Retroviral cDNA Integration: Stimulation by HMG I Family Proteins

ABSTRACT To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.

Alterations of the Tumor Suppressor Gene Parkin in Non-Small Cell Lung Cancer

Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the common fragile site FRA6E on human chromosome 6q25-q27, is associated with a frequent loss of heterozygosity and altered expression in breast and ovarian carcinomas. In addition, homozygous deletions of exon 2 creating deleterious truncations of the Parkin transcript were observed in the lung adenocarcinoma cell lines Calu-3 and H-1573, suggesting that the loss of this locus and the resulting changes in its expression are involved in the development of these tumors. Experimental Design: We examined 20 paired normal and non-small cell lung cancer samples for the presence of Parkin alterations in the coding sequence and changes in gene expression. We also restored gene expression in the Parkin-deficient lung carcinoma cell line H460 by use of a recombinant lentivirus containing the wild-type Parkin cDNA. Results: Loss of heterozygosity analysis identified a common region of loss in the Parkin/FRA6E locus with the highest frequency for the intragenic marker D6S1599 (45%), and semi-quantitative reverse transcription-PCR revealed reduced expression in 3 of 9 (33%) lung tumors. Although we did not observe any in vitro changes in cell proliferation or cell cycle, ectopic Parkin expression had the ability to reduce in vivo tumorigenicity in nude mice. Conclusion: These data suggest that Parkin is a tumor suppressor gene whose inactivation may play an important role in non-small cell lung cancer tumorigenesis.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

The BCSC-1 locus at chromosome 11q23-q24 is a candidate tumor suppressor gene

Frequent allelic loss at human chromosome 11q23-q24 occurs in a wide variety of cancers, suggesting that this region may harbor a tumor suppressor gene. By constructing a physical map of the LOH11CR2 minimal region of loss on 11q23-q24 associated with lung and breast carcinomas, we were able to clone a hereditary translocation, t(11;12)(q23;q24), in a patient with early-onset breast cancer and family history of cancer. The breakpoint was found within 6 kb of the BCSC-1 candidate tumor suppressor gene located in the LOH11CR2 region whereas additional loss of heterozygosity (LOH) analysis in breast and ovarian tumors, including that of the patient with the t(11;12)(q23;q24), implicated the BCSC-1 locus as the primary target of deletion. Northern analysis of the BCSC-1 mRNA revealed a lack of expression in 33 of 41 (80%) tumor cell lines, and its ectopic expression led to the suppression of colony formation in vitro and tumorigenicity in vivo. These data suggest that BCSC-1 may exert a tumor suppressor activity and is a likely target of the LOH observed on 11q23-q24 in cancer.

siRNA Screening of a Targeted Library of DNA Repair Factors in HIV Infection Reveals a Role for Base Excision Repair in HIV Integration

Host DNA repair enzymes have long been assumed to play a role in HIV replication, and many different DNA repair factors have been associated with HIV. In order to identify DNA repair pathways required for HIV infection, we conducted a targeted siRNA screen using 232 siRNA pools for genes associated with DNA repair. Mapping the genes targeted by effective siRNA pools to well-defined DNA repair pathways revealed that many of the siRNAs targeting enzymes associated with the short patch base excision repair (BER) pathway reduced HIV infection. For six siRNA pools targeting BER enzymes, the negative effect of mRNA knockdown was rescued by expression of the corresponding cDNA, validating the importance of the gene in HIV replication. Additionally, mouse embryo fibroblasts (MEFs) lacking expression of specific BER enzymes had decreased transduction by HIV-based retroviral vectors. Examining the role BER enzymes play in HIV infection suggests a role for the BER pathway in HIV integration.

Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance.

The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration

An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.