Three Pol X family members have been linked to nonhomologous end joining (NHEJ) in mammals. Template-independent TdT promotes diversity during NHEJ-dependent repair of V(D)J recombination ...intermediates, but the roles of the template-dependent polymerases μ and λ in NHEJ remain unclear. We show here that pol μ and pol λ are similarly recruited by NHEJ factors to fill gaps when ends have partially complementary overhangs, suggesting equivalent roles promoting accuracy in NHEJ. However, only pol μ promotes accuracy during immunoglobulin kappa recombination. This distinctive in vivo role correlates with the TdT-like ability of pol μ, but not pol λ, to act when primer termini lack complementary bases in the template strand. However, unlike TdT, synthesis by pol μ in this context is primarily instructed by a template from another DNA molecule. This apparent gradient of template dependence is largely attributable to a small structural element that is present but different in all three polymerases.
Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks. A key feature of biological sources of strand breaks is associated ...nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites). At single-strand breaks, 5′-terminal abasic sites are excised by the 5′-deoxyribose-5-phosphate (5′-dRP) lyase activity of DNA polymerase (pol ): here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5′-dRP/AP lyase activity. Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping to distinguish this pathway from otherwise robust alternative NHEJ pathways. The NHEJ factor Ku can be identified as an effective 5′-dRP/AP lyase. In a similar manner to other lyases, Ku nicks DNA 3′ of an abasic site by a mechanism involving a Schiff-base covalent intermediate with the abasic site. We show by using cell extracts that Ku is essential for the efficient removal of AP sites near double-strand breaks and, consistent with this result, that joining of such breaks is specifically decreased in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA polymerase μ is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol μ's ability ...to promote NHEJ of ends with noncomplementary 3′ overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol μ in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol μ is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol μ is less able to synthesize across longer gaps, arguing pol μ must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol μ mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol μ activity during NHEJ of noncomplementary ends can thus be primarily linked to pol μ's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction.
Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA ...binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis ...and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced by ionizing radiation. Hydroxyurea additionally reduces the frequency of irradiated cells with a marker for an early step in HR, Rad51 foci, consistent with reduced initiation of HR under these conditions. Conversely, promotion of ribonucleotide reductase activity protects NHEJ-deficient cells from ionizing radiation. Importantly, promotion of ribonucleotide reductase activity also increases usage of HR in cells proficient in both NHEJ and HR at a targeted chromosome break. Activity of ribonucleotide reductase is thus an important factor in determining how mammalian cells repair broken chromosomes. This may explain in part why G1/G0 cells, which have reduced ribonucleotide reductase activity, rely more on NHEJ for DSB repair.
Three of the four family X polymerases, DNA polymerase λ, DNA polymerase μ, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in ...this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase λ, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase λ BRCT domain reveals structural similarity to the BRCT domains of polymerase μ and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol λ plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol μ, Arg43. In contrast, the hydrophobic Leu60 residue of Pol λ contributes less significantly to binding than the structurally homologous Phe46 residue of Pol μ. A third leucine residue involved in the binding and activity of Pol μ, is nonconservatively replaced by a glutamine in Pol λ (Gln64) and, based on binding and activity data, is apparently unimportant for Pol λ interactions with the NHEJ complex. In conclusion, both the structure of the Pol λ BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol μ and TdT.
DNA polymerases β and η are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both ...polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin−GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated. In this study, we performed a steady-state kinetic analysis of the steps required for fixation of dTTP misinsertion during translesion synthesis past cisplatin- and oxaliplatin−GG adducts by pol β and pol η. The efficiency of translesion synthesis by pol η past Pt−GG adducts was very similar to that observed for this polymerase when the template contains thymine−thymine dimers. This finding suggested that pol η could play a role in translesion synthesis past platinum−GG adducts in vivo. On the other hand, translesion synthesis past platinum−GG adducts by pol β was much less efficient. Translesion synthesis by pol η is likely to be predominantly error-free, since the probability of correct insertion and extension by pol η was 1000−2000-fold greater than the probability of incorrect insertion and extension. Our results also indicated that for pol η the frequency of misincorporation is the same across from the 3‘G and the 5‘G of the platinum−GG adducts for both cisplatin and oxaliplatin adducts. On the other hand, pol β is more likely to misinsert at the 3‘G of the adducts and misinsertion occurs at higher frequency for oxaliplatin−GG than for cisplatin−GG adducts.
DNA polymerase μ (pol μ) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase β (pol β) and terminal deoxynucleotidyl transferase ...(TdT). Because pol β has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol μ to bypass these lesions. Pol μ bypassed CP and OX adducts with an efficiency of 14−35% compared to chain elongation on undamaged DNA, which is second only to pol η in terms of bypass efficiency. The relative ability of pol μ to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol μ has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol μ to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol μ was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol μ may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream “acceptor” and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.
The solution structure and dynamics of the BRCT domain from human DNA polymerase mu, implicated in repair of chromosome breaks by nonhomologous end joining (NHEJ), has been determined using NMR ...methods. BRCT domains are typically involved in protein-protein interactions between factors required for the cellular response to DNA damage. The pol mu BRCT domain is atypical in that, unlike other reported BRCT structures, the pol mu BRCT is neither part of a tandem grouping, nor does it appear to form stable homodimers. Although the sequence of the pol mu BRCT domain has some unique characteristics, particularly the presence of >10% proline residues, it forms the characteristic alphabetaalpha sandwich, in which three alpha helices are arrayed around a central four-stranded beta-sheet. The structure of helix alpha1 is characterized by two solvent-exposed hydrophobic residues, F46 and L50, suggesting that this element may play a role in mediating interactions of pol mu with other proteins. Consistent with this argument, mutation of these residues, as well as the proximal, conserved residue R43, specifically blocked the ability of pol mu to efficiently work together with NHEJ factors Ku and XRCC4-ligase IV to join noncomplementary ends together in vitro. The structural, dynamic, and biochemical evidence reported here identifies a functional surface in the pol mu BRCT domain critical for promoting assembly and activity of the NHEJ machinery. Further, the similarity between the interaction regions of the BRCT domains of pol mu and TdT support the conclusion that they participate in NHEJ as alternate polymerases.
Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA ...binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair. PUBLICATION ABSTRACT
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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