Showing papers on "DNA clamp published in 2007"

Structural basis for DNA duplex separation by a superfamily-2 helicase.

Abstract: To reveal the mechanism of processive strand separation by superfamily-2 (SF2) 3′→5′ helicases, we determined apo and DNA-bound crystal structures of archaeal Hel308, a helicase that unwinds lagging strands and is related to human DNA polymerase θ Our structure captures the duplex-unwinding reaction, shows that initial strand separation does not require ATP and identifies a prominent β-hairpin loop as the unwinding element Similar loops in hepatitis C virus NS3 helicase and RNA-decay factors support the idea that this duplex-unwinding mechanism is applicable to a broad subset of SF2 helicases Comparison with ATP-bound SF2 enzymes suggests that ATP promotes processive unwinding of 1 base pair by ratchet-like transport of the 3′ product strand Our results provide a first structural framework for strand separation by processive SF2 3′→5′ helicases and reveal important mechanistic differences from SF1 helicases

Human immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration

Abstract: Encapsidation of host restriction factor APOBEC3G (A3G) into vif-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.

Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

Abstract: Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks.

Abstract: DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.

8-oxo-guanine bypass by human DNA polymerases in the presence of auxiliary proteins

Abstract: Specialized DNA polymerases (DNA pols) are required for lesion bypass in human cells. Auxiliary factors have an important, but so far poorly understood, role. Here we analyse the effects of human proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) on six different human DNA pols--belonging to the B, Y and X classes--during in vitro bypass of different lesions. The mutagenic lesion 8-oxo-guanine (8-oxo-G) has high miscoding potential. A major and specific effect was found for 8-oxo-G bypass with DNA pols lambda and eta. PCNA and RP-A allowed correct incorporation of dCTP opposite a 8-oxo-G template 1,200-fold more efficiently than the incorrect dATP by DNA pol lambda, and 68-fold by DNA pol eta, respectively. Experiments with DNA-pol-lambda-null cell extracts suggested an important role for DNA pol lambda. On the other hand, DNA pol iota, together with DNA pols alpha, delta and beta, showed a much lower correct bypass efficiency. Our findings show the existence of an accurate mechanism to reduce the deleterious consequences of oxidative damage and, in addition, point to an important role for PCNA and RP-A in determining a functional hierarchy among different DNA pols in lesion bypass.

Bypass of DNA Lesions Generated During Anticancer Treatment with Cisplatin by DNA Polymerase η

Abstract: DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis-syn thymine dimers. We present structural and biochemical analysis of how Pol eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.

Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

Abstract: Hepatitis B virus (HBV) contains a small, partially double-stranded, relaxed circular (RC) DNA genome. RC DNA needs to be converted to covalently closed circular (CCC) DNA, which serves as the template for all viral RNA transcription. As a first step toward understanding how CCC DNA is formed, we analyzed the viral and host factors that may be involved in CCC DNA formation, using transient and stable DNA transfections of HBV and the related avian hepadnavirus, duck hepatitis B virus (DHBV). Our results show that HBV CCC DNA formed in hepatoma cells was derived predominantly from RC DNA with a precise junction sequence. In contrast to that of DHBV, HBV CCC DNA formation in cultured cells was accompanied by the accumulation of a RC DNA species from which the covalently attached viral reverse transcriptase (RT) protein was removed (protein-free or PF-RC DNA). Furthermore, whereas envelope deficiency led to increased CCC DNA formation in DHBV, it resulted mainly in increased PF-RC, but not CCC, DNA in HBV, suggesting that the envelope protein(s) may negatively regulate a step in CCC DNA formation that precedes deproteination in both HBV and DHBV. Interestingly, PF-RC DNA, in contrast to RT-linked RC DNA, contained, almost exclusively, mature plus-strand DNA, suggesting that the RT protein was removed preferentially from mature RC DNA.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

Abstract: We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage.

Abstract: All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co-ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at specific sites on the chromosome termed origins of replication, and proceeds bidirectionally to elongate and duplicate the chromosome. These two processes, initiation and elongation, therefore determine the total rate of DNA synthesis in the cell. In Escherichia coli, initiation depends on the DnaA protein while elongation depends on a multiprotein replication factory that incorporates deoxyribonucleotides (dNTPs) into the growing DNA chain. The enzyme ribonucleotide reductase (RNR) is universally responsible for synthesizing the necessary dNTPs. In this review we examine the role RNR plays in regulating the total rate of DNA synthesis in E. coli and, hence, in maintaining constant DNA/cell mass ratios during normal growth and under conditions of DNA stress.

Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

Abstract: The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.

Replication Origin Recognition and Deformation by a Heterodimeric Archaeal Orc1 Complex

Abstract: The faithful duplication of genetic material depends on essential DNA replication initiation factors. Cellular initiators form higher-order assemblies on replication origins, using adenosine triphosphate (ATP) to locally remodel duplex DNA and facilitate proper loading of synthetic replisomal components. To better understand initiator function, we determined the 3.4 angstrom-resolution structure of an archaeal Cdc6/Orc1 heterodimer bound to origin DNA. The structure demonstrates that, in addition to conventional DNA binding elements, initiators use their AAA+ ATPase domains to recognize origin DNA. Together these interactions establish the polarity of initiator assembly on the origin and induce substantial distortions into origin DNA strands. Biochemical and comparative analyses indicate that AAA+/DNA contacts observed in the structure are dynamic and evolutionarily conserved, suggesting that the complex forms a core component of the basal initiation machinery.

Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs.

Abstract: Recognition of DNA double-strand breaks during non-homologous end joining is carried out by the Ku70–Ku80 protein, a 150 kDa heterodimer that recruits the DNA repair kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to the lesion. The atomic structure of a truncated Ku70–Ku80 was determined; however, the subunit-specific carboxy-terminal domain of Ku80—essential for binding to DNA-PKcs—was determined only in isolation, and the C-terminal domain of Ku70 was not resolved in its DNA-bound conformation. Both regions are conserved and mediate protein–protein interactions specific to mammals. Here, we reconstruct the three-dimensional structure of the human full-length Ku70–Ku80 dimer at 25 A resolution, alone and in complex with DNA, by using single-particle electron microscopy. We map the C-terminal regions of both subunits, and their conformational changes after DNA and DNA-PKcs binding to define a molecular model of the functions of these domains during DNA repair in the context of full-length Ku70–Ku80 protein.

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross‐Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

Abstract: Single-step aqueous cross-coupling reactions of nucleobase-halogenated 2'-deoxynucleosides (8-bromo-2'-deoxyadenosine, 7-iodo-7-deaza-2'-deoxyadenosine, or 5-iodo-2'-deoxy-uridine) or their 5'-triphosphates with 4-boronophenylalanine or 4-ethynylphenylalanine have been developed and used for efficient synthesis of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8-substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7-substituted 7-deazaadenine and 5-substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two-step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.

A Water-Mediated and Substrate-Assisted Catalytic Mechanism for Sulfolobus solfataricus DNA Polymerase IV

Abstract: DNA polymerases are enzymes responsible for the synthesis of DNA from nucleotides. Understanding their molecular fundamentals is a prerequisite for elucidating their aberrant activities in diseases such as cancer. Here we have carried out ab initio quantum mechanical/molecular mechanical (QM/MM) studies on the nucleotidyl-transfer reaction catalyzed by the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus, with template guanine and Watson−Crick paired dCTP as the nascent base pair. The results suggested a novel water-mediated and substrate-assisted (WMSA) mechanism: the initial proton transfer to the α-phosphate of the substrate via a bridging crystal water molecule is the rate-limiting step, the nucleotidyl-transfer step is associative with a metastable pentacovalent phosphorane intermediate, and the pyrophosphate leaving is facilitated by a highly coordinated proton relay mechanism through mediation of water which neutralizes the evolving negative charge. The conserved carboxylates, w...

Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules

Abstract: Using total internal reflection fluorescence microscopy, we directly visualize in real-time, the 1D Brownian motion and transcription elongation of T7 RNA polymerase along aligned DNA molecules bound to substrates by molecular combing. We fluorescently label T7 RNA polymerase with antibodies and use flow to convect them orthogonally to the DNA alignment direction, permitting observation and estimation of the protein diffusivity along the DNA at the single-molecule level. Our observations suggest that the 1D diffusion coefficient varies from molecule to molecule over the range 6.1 x 10(-11) cm2/s to 4.3 x 10(-9) cm2/s. We also observe binding and transcription by T7 RNA polymerases on single combed T7 DNA molecules with an apparent association rate of 1.6 microM(-1)s(-1). From the measured dependence of the rate of transcription on concentration of nucleotide triphosphate, we infer that the combed DNA molecules capable of interacting with proteins are under an average tension of 25 pN.

A common topology for bacterial and eukaryotic transcription initiation

Abstract: DNA supercoiling is a major regulator of transcription in bacteria. Negative supercoiling acts both by promoting the formation of nucleoprotein structures containing wrapped DNA and by altering the twist of DNA. The latter affects the initiation of transcription by RNA polymerase as well as recombination processes. Here, we argue that although bacteria and eukaryotes differ in their mode of packaging DNA supercoils, increases in DNA twist are associated with chromatin folding and transcriptional silencing in both. Conversely, decreases in DNA twist are associated with chromatin unfolding and the acquisition of transcriptional competence. In other words, at the most fundamental level, the principles of genetic regulation are common to all organisms. The apparent differences in the details of regulation probably represent alternative methods of fine-tuning similar underlying processes.

Electrochemical quantification of single-nucleotide polymorphisms using nanoparticle probes.

Abstract: We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol of mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction preamplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.

Structural insight into the substrate specificity of DNA Polymerase |[mu]|

Abstract: DNA polymerase μ (Pol μ) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol μ's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol μ bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol μ that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol μ from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3′ ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.

Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.

Abstract: In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases η and ζ and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.

Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo.

Abstract: Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polκ, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.

Reactive oxygen-mediated damage to a human DNA replication and repair protein

Abstract: Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[a]pyrene dG adduct in DNA through template misalignment.

Abstract: Erroneous replication of lesions in DNA by DNA polymerases leads to elevated mutagenesis. To understand the molecular basis of DNA damage-induced mutagenesis, we have determined the x-ray structures of the Y-family polymerase, Dpo4, in complex with a DNA substrate containing a bulky DNA lesion and incoming nucleotides. The DNA lesion is derived from an environmentally widespread carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The potent carcinogen BP is metabolized to diol epoxides that form covalent adducts with cellular DNA. In the present study, the major BP diol epoxide adduct in DNA, BP-N2-deoxyguanosine (BP–dG), was placed at a template–primer junction. Three ternary complexes reveal replication blockage, extension past a mismatched lesion, and a −1 frameshift mutation. In the productive structures, the bulky adduct is flipped/looped out of the DNA helix into a structural gap between the little finger and core domains. Sequestering of the hydrophobic BP adduct in this new substrate-binding site permits the DNA to exhibit normal geometry for primer extension. Extrusion of the lesion by template misalignment allows the base 5′ to the adduct to serve as the template, resulting in a −1 frameshift. Subsequent strand realignment produces a mismatched base opposite the lesion. These structural observations, in combination with replication and mutagenesis data, suggest a model in which the additional substrate-binding site stabilizes the extrahelical nucleotide for lesion bypass and generation of base substitutions and −1 frameshift mutations.