Biomedical subjects

Paul R Andreassen

Publications and source records attributed to Paul R Andreassen.

14 recordsLinked to original sources

PubMed2006

DNA damage responses and their many interactions with the replication fork.

The cellular response to DNA damage is composed of cell cycle checkpoint and DNA repair mechanisms that serve to ensure proper replication of the genome prior to cell division. The function of the DNA damage response during DNA replication in S-phase is critical to this process. Recent evidence has suggested a number of interrelationships of DNA replication and cellular DNA damage responses. These include S-phase checkpoints which suppress replication initiation or elongation in response to DNA damage. Also, many components of the DNA damage response are required either for the stabilization of, or for restarting, stalled replication forks. Further, translesion synthesis permits DNA replication to proceed in the presence of DNA damage and can be coordinated with subsequent repair by homologous recombination (HR). Finally, cohesion of sister chromatids is established coincident with DNA replication and is required for subsequent DNA repair by homologous recombination. Here we review these processes, all of which occur at, or are related to, the advancing replication fork. We speculate that these multiple interdependencies of DNA replication and DNA damage responses integrate the many steps necessary to ensure accurate duplication of the genome.

Animals↗

PubMed2006

Assessing the link between BACH1 and BRCA1 in the FA pathway.

The BACH1 helicase was initially identified by its direct binding to BRCA1 and, thus, was linked to hereditary breast cancer. More recently, BACH1 was identified as the gene defective in the J complementation group of Fanconi anemia (FA). FA is a multigenetic disorder characterized by cellular sensitivity to crosslinkers and chromosome instability. Because FANCD2 monoubiquitination is intact in BACH1 deficient cells, BACH1 appears to act downstream in the FA pathway akin to BRCA2/FANCD1. Interestingly, while BRCA1 has various interactions with FA proteins it has not been identified as an FA gene. As the race to uncover the last few unknown FA complementation groups comes to an end, future work will be required to uncover how these gene products function to combat the effects of DNA damage and maintain genomic stability. In particular, it remains elusive whether BRCA1 is functionally linked to the FA pathway through its interaction with BACH1/FANCJ. This review focuses on a model for the connection of BRCA1 to BACH1 in the FA pathway. We predict that BRCA1 regulates the BACH1 helicase activity to coordinate the timely displacement of Rad51 from nucleofilaments, promoting error free repair and ultimately maintaining chromosomal integrity.

Animals↗

PubMed2005

BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ.

We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.

Basic-Leucine Zipper Transcription Factors↗

PubMed2004

Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin.

DNA damage activates the monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, resulting in the assembly of FANCD2 nuclear foci. In the current study, we characterize structural features of FANCD2 required for this intranuclear translocation. We have previously identified 2 normal mRNA splice variants of FANCD2, one containing exon 44 sequence at the 3' end (FANCD2-44) and one containing exon 43 sequence (FANCD2-43). The 2 predicted FANCD2 proteins differ in their carboxy terminal 24 amino acids. In stably transfected FANCD2(-/-) fibroblasts, FANCD2-44 and FANCD2-43 proteins were monoubiquitinated on K561. Only FANCD2-44 corrected the mitomycin C (MMC) sensitivity of the transfected cells. We find that monoubiquitinated FANCD2-44 was translocated from the soluble nuclear compartment into chromatin. A mutant form of FANCD2-44 (FANCD2-K561R) was not monoubiquitinated and failed to bind chromatin. A truncated FANCD2 protein (Exon44-T), lacking the carboxy terminal 24 amino acids encoded by exon 44 but retaining K561, and another mutant FANCD2 protein, with a single amino acid substitution at a conserved residue within the C-terminal 24 amino acids (D1428A), were monoubiquitinated. Both mutants were targeted to chromatin but failed to correct MMC sensitivity. Taken together, our results indicate that monoubiquitination of FANCD2 regulates chromatin binding and that D1428 within the carboxy terminal acidic sequence encoded by exon 44 is independently required for functional complementation of FA-D2 cells. We hypothesize that the carboxy terminus of FANCD2-44 plays a critical role in sensing or repairing DNA damage.

Cell Cycle↗

PubMed2004

Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase.

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.

Cell Line, Tumor↗

PubMed2004

ATR couples FANCD2 monoubiquitination to the DNA-damage response.

Fanconi anemia (FA) is a multigenic autosomal recessive cancer susceptibility syndrome. The FA pathway regulates the monoubiquitination of FANCD2 and the assembly of damage-associated FANCD2 nuclear foci. How FANCD2 monoubiquitination is coupled to the DNA-damage response has remained undetermined. Here, we demonstrate that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination. Deficiency of ATR function, either in Seckel syndrome, which clinically resembles Fanconi anemia, or by siRNA silencing, results in the formation of radial chromosomes in response to the DNA cross-linker, mitomycin C (MMC), thus mimicking the chromosome instability of FA cells.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2(-/-) cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2(-/-) cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.

BRCA2 Protein↗

PubMed2004

Analysis of the spindle-assembly checkpoint in HeLa cells.

The spindle-assembly checkpoint involves signaling at kinetochores, which leads to the arrest of mitotic progression in the absence of microtubule attachment or spindle tension. Here, we detail procedures for the analysis of the spindle-assembly checkpoint in adherent mammalian cells. These techniques focus on pharmacological approaches and immunofluorescence microscopy to verify the state of spindle assembly, kinetochore attachment of microtubules and spindle tension, chromosome positioning, and kinetochore signaling by the Mad2 or Bub1 checkpoint proteins. We also describe a bi-parameter flow cytometric assay, using either MPM-2 or anti-phospho-(Ser10)-histone H3 antibodies, for quantitating mitotic cells.

Carrier Proteins↗

PubMed2003

G2 and spindle assembly checkpoint adaptation, and tetraploidy arrest: implications for intrinsic and chemically induced genomic instability.

While checkpoints that act in S-phase are essential to the maintenance of genomic stability, these checkpoints do not act alone. Additionally, G2 DNA damage checkpoints, the spindle assembly checkpoint, and a post-mitotic G1 tetraploidy checkpoint act subsequent to DNA replication to ensure genetic fidelity in cell division. In this review, we will examine how these checkpoints cooperate in the maintenance of genomic stability in response to either DNA damage or cytoskeletal disruption. Since the G2 and spindle assembly checkpoints are subject to adaptation, we will discuss how the G1 tetraploidy checkpoint acts in concert with these checkpoints to mediate stable arrest. We will also probe the relationship of these checkpoints by exploring common features of their regulation. Finally, the consequences of malfunction of these checkpoints for both intrinsic and chemically induced genomic instability will be examined. Among these consequences are aneuploidization, extranumerary centrosomes, and micronucleation.

Cell Cycle Proteins↗

PubMed2003

Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53.

p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

Actins↗

PubMed2003

G1 tetraploidy checkpoint and the suppression of tumorigenesis.

Checkpoints suppress improper cell cycle progression to ensure that cells maintain the integrity of their genome. During mitosis, a metaphase checkpoint requires the integration of all chromosomes into a metaphase array in the mitotic spindle prior to mitotic exit. Still, mitotic errors occur in mammalian cells with a relatively high frequency. Metaphase represents the last point of control in mitosis. Once the cell commits to anaphase there are no checkpoints to sense segregation defects. In this context, we will explore our recent finding that non-transformed mammalian cells have a checkpoint that acts subsequent to mitotic errors to block the proliferation of cells that have entered G1 with tetraploid status. This arrest is dependent upon both p53 and pRb, and may represent an important function of both p53 and pRb as tumor suppressors. Further, we discuss the possibility that this mechanism may similarly impose G1 arrest in cells that become aneuploid through errors in mitosis.

Aneuploidy↗

PubMed2003

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression.

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

Amino Acid Sequence↗

PubMed2002

S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51.

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and defective cell-cycle progression. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common pathway. DNA damage activates this pathway, leading to monoubiquitination of the downstream FANCD2 protein and targeting to nuclear foci containing BRCA1. In the current study, we demonstrate that FANCD2 also undergoes monoubiquitination during S phase of the cell cycle. Monoubiquitinated FANCD2 colocalizes with BRCA1 and RAD51 in S-phase-specific nuclear foci. Monoubiquitination of FANCD2 is required for normal cell-cycle progression following cellular exposure to mitomycin C. Our data indicate that the monoubiquitination of FANCD2 is highly regulated, and they suggest that FANCD2/BRCA1 complexes and FANCD2/RAD51 complexes participate in an S-phase-specific cellular process, such as DNA repair by homologous recombination.

BRCA1 Protein↗

PubMed2002

Convergence of the fanconi anemia and ataxia telangiectasia signaling pathways.

Fanconi anemia (FA) and ataxia telangiectasia (AT) are clinically distinct autosomal recessive disorders characterized by spontaneous chromosome breakage and hematological cancers. FA cells are hypersensitive to mitomycin C (MMC), while AT cells are hypersensitive to ionizing radiation (IR). Here, we identify the Fanconi anemia protein, FANCD2, as a link between the FA and ATM damage response pathways. ATM phosphorylates FANCD2 on serine 222 in vitro. This site is also phosphorylated in vivo in an ATM-dependent manner following IR. Phosphorylation of FANCD2 is required for activation of an S phase checkpoint. The ATM-dependent phosphorylation of FANCD2 on S222 and the FA pathway-dependent monoubiquitination of FANCD2 on K561 are independent posttranslational modifications regulating discrete cellular signaling pathways. Biallelic disruption of FANCD2 results in both MMC and IR hypersensitivity.

Ataxia Telangiectasia↗