Genetic Witness Aronson, Jay D
2007, 20071011, 2007-10-11
eBook
When DNA profiling was first introduced into the American legal system in 1987, it was heralded as a technology that would revolutionize law enforcement. As an investigative tool, it has lived up to ...much of this hype-it is regularly used to track down unknown criminals, put murderers and rapists behind bars, and exonerate the innocent.Yet, this promise took ten turbulent years to be fulfilled. In Genetic Witness, Jay D. Aronson uncovers the dramatic early history of DNA profiling that has been obscured by the technique's recent success. He demonstrates that robust quality control and quality assurance measures were initially nonexistent, interpretation of test results was based more on assumption than empirical evidence, and the technique was susceptible to error at every stage. Most of these issues came to light only through defense challenges to what prosecutors claimed to be an infallible technology. Although this process was fraught with controversy, inefficiency, and personal antagonism, the quality of DNA evidence improved dramatically as a result. Aronson argues, however, that the dream of a perfect identification technology remains unrealized.
The number of DNA polymerases identified in each organism has mushroomed in the past two decades. Most newly found DNA polymerases specialize in translesion synthesis and DNA repair instead of ...replication. Although intrinsic error rates are higher for translesion and repair polymerases than for replicative polymerases, the specialized polymerases increase genome stability and reduce tumorigenesis. Reflecting the numerous types of DNA lesions and variations of broken DNA ends, translesion and repair polymerases differ in structure, mechanism, and function. Here, we review the unique and general features of polymerases specialized in lesion bypass, as well as in gap-filling and end-joining synthesis.
Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including ...cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.
Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, ...Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.
The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase ...activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.
BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during ...unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.
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•ssDNA gaps arise in BRCA1 mutant cancer cells due to PRIMPOL-mediated repriming•BRCA1/2 deficiency leads to mutagenic ssDNA gap repair by REV1-Polζ-dependent TLS•Targeted REV1-Polζ inhibition shows enhanced toxicity in HR-deficient cancer cells•ssDNA gaps formed by SMUG1 and PRIMPOL mediate the toxicity of REV1-Polζ inhibition
Taglialatela et al. report that homologous recombination (HR)-deficient cancer cells, such as BRCA1/2 mutant cells, display increased reliance on error-prone translesion synthesis (TLS) for the repair of ssDNA gaps arising spontaneously during DNA replication. TLS inhibition shows exquisite toxicity in BRCA1/2-deficient cancer cells, providing the basis for alternative therapies against BRCA1/2 mutant tumors.
DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have ...identified strand-separating “pin” structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein–DNA cross-linking, we localized the putative pin (a β-hairpin within a zinc-binding domain in PriA) near the ssDNA–dsDNA junction of the lagging strand in a PriA–DNA replication fork complex. Removal of residues at the tip of the β-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA β-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted β-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro. These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA ...synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
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•HLTF mediates fork reversal in vivo, associated with DSB formation•HLTF prevents unrestrained replication driven by PRIMPOL or the TLS protein REV1•Unrestrained DNA synthesis promotes S phase progression under replication stress•HLTF loss increases cellular resistance to replication stress and ATR inhibition
Under replication stress, cells deficient in the fork remodeler HLTF fail to slow DNA replication. Here, Bai et al. report that, when HLTF is disrupted, replication is completed by alternative PRIMPOL- or REV1-dependent mechanisms. Both replication modes are potentially mutagenic and lead to replication stress resistance.
Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA ...synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap ...filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
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•Two temporally distinct pathways fill ssDNA gaps in human cells•RAD18, PCNA monoubiquitination, and REV1-POLζ promote gap filling in G2•UBC13, RAD51, and REV1-POLζ promote gap filling in S•BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity in S and G2
PRIMPOL generates ssDNA gaps, which must be filled to maintain genome stability. Tirman et al. show that two distinct pathways fill ssDNA gaps throughout the cell cycle in human cells and that BRCA proteins promote gap filling by limiting MRE11 activity. Disruption of these pathways enhances genome instability and chemosensitivity.
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