Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Characterization of the human and mouse WRN 3'-->5' exonuclease

Werner’s syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3′→5′ DNA helicase activity. We recently showed that hWRN is also a 3′→5′ exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3′ recessed strands of double-stranded DNA or a DNA–RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3′ protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3′ recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3′→5′ exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.     

Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen

The Apn2 protein of Saccharomyces cerevisiae contains 3'-->5' exonuclease and 3'-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3'-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.     

Oligonucleotide N3'-->P5' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3'-->P5' phosphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with complementary native phosphodiester DNA and exceptionally stable duplexes with RNA strands. Increases in duplex melting temperature, deltaTm, relatively to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modified nucleoside. Moreover, the phosphoramidate compounds form extremely stable triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of these triplexes usually exceed that of the isosequential phosphodiester counterparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nucleases both in vitro and in vivo. Oligonucleotide phosphoramidates apparently are cell permeable, and they have a good bioavailability and biodistribution, while being non-toxic in mice at therapeutically relevant doses. Duplexes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these compounds exerted high sequence-specific antisense activity in various cell lines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biological activity of these compounds relative to RNase H inducing oligonucleotides. Experimental results also indicate that oligonucleotide phosphoramidates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidate double stranded complexes can structurally mimic native RNA complexes, which could be efficiently and specifically recognized by the RNA binding proteins, such as HIV-1 Rev and Tat.     

ARE-mRNA degradation requires the 5'-3' decay pathway

As an important mode of suppressing gene expression, messenger RNAs containing an AU-rich element (ARE) in the 3' untranslated region are rapidly degraded in the cytoplasm. ARE-mediated mRNA decay (AMD) is initiated by deadenylation, and in vitro studies have indicated that subsequent degradation occurs in the 3'-5' direction through a complex of exonucleases termed the exosome. An alternative pathway of mRNA degradation occurs at processing bodies, cytoplasmic foci that contain decapping enzymes, the 5'-3' exonuclease Xrn1 and the Lsm1-7 heptamer. To determine which of the two pathways is important for AMD in live cells, we targeted components of both pathways using short interfering RNA in human HT1080 cells. We show that Xrn1 and Lsm1 are essential for AMD. On the other side, out of three exosome components tested, only knockdown of PmScl-75 caused a strong inhibition of AMD. Our results show that mammalian cells, similar to yeast, require the 5'-3' Xrn1 pathway to degrade ARE-mRNAs.     

Arrayed primer extension on in situ synthesized 5'-->3' oligonucleotides in microchannels

Arrays of oligonucleotides synthesized in the 5'-->3' direction have potential benefit in several areas of life sciences research because the free 3'-end can be modified by enzymatic reactions. A Geniom One instrument (febit biomed GmbH, Germany), with integrated chip fabrication, multiplex primer extension, fluorescence imaging, and data analysis, was evaluated for studies of genomic variations. Microchannels used for the array synthesis in Geniom One were not optimized before for the APEX method and, as preliminary experiments demonstrated in this study, the signals were strongly affected by the speed of the process inside reaction channels. Using the two-compartment model (TCM), target binding to feature were quantitatively analyzed, revealing profound mass-transport limitations in the observed kinetics and enabling us to draw a series of physicochemical conclusions of the optimal set-up for the APEX reaction. Some kinetically relevant parameters such as target concentration, reaction time, and temperature were comprehensively analyzed. Finally, we applied the arrays and methods in a proof-of-principle experiment where 36 individuals were typed with 900 oligonucleotide probes (sense and antisense primers for 450 markers), using the ABCR gene as a test system. A new DNA analysis method for studies of genomic variation was developed using this all-in-one platform.     

Cyclic 3',5'-adenosine monophosphate stimulates trehalose degradation in baker's yeast

The levels of cyclic 3′,5′-AMP and trehalose, as well as the specific activity of the trehalase have been investigated in cells of baker's yeast ($\text{Saccharomyces cerevisiae}$) during the lag phase preceding growth. During the first few minutes a substantial increase in the intracellular concentration of cyclic 3′,5′-AMP was observed, followed by a 6–8 fold increase in trehalase activity concomitant with the rapid degradation of trehalose. Cell free extracts prepared from resting yeast were shown to contain a cryptic trehalase, which under physiological conditions could be activated by cyclic 3′,5′-AMP to the same degree as $\text{in vivo}$. These observations suggest that in the lag phase of growth, the level of trehalose in baker's yeast is under control of a system, regulated by the level of cyclic 3′,5′-AMP.     

A new approach to oligonucleotide N3'-->P5' phosphoramidate building blocks

A new synthetic approach to 5-phosphoramidites of 3'-aminonucleosides was developed. The methodology relies upon the use of 3'-amino-2',3'-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2-3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3'-aminonucleoside sugar and base moieties.     

3'-phosphodiesterase and 3'-->5' exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage

In Saccharomyces cerevisiae, the AP endonucleases encoded by the APN1 and APN2 genes provide alternate pathways for the removal of abasic sites. Oxidative DNA-damaging agents, such as H(2)O(2), produce DNA strand breaks which contain 3'-phosphate or 3'-phosphoglycolate termini. Such 3' termini are inhibitory to synthesis by DNA polymerases. Here, we show that purified yeast Apn2 protein contains 3'-phosphodiesterase and 3'-->5' exonuclease activities, and mutation of the active-site residue Glu59 to Ala in Apn2 inactivates both these activities. Consistent with these biochemical observations, genetic studies indicate the involvement of APN2 in the repair of H(2)O(2)-induced DNA damage in a pathway alternate to APN1, and the Ala59 mutation inactivates this function of Apn2. From these results, we conclude that the ability of Apn2 to remove 3'-end groups from DNA is paramount for the repair of strand breaks arising from the reaction of DNA with reactive oxygen species.     

The correcting role of autonomous 3'-->5' exonucleases in mammalian multienzyme DNA polymerase complexes

A study was made of the correcting role of autonomous 3'-->5' exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage psi X174 amber3 or M13mp2 primer-templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The mistake rate of the hepatocytic complex was estimated at 3 x 10(-6) with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3'-->5' exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (epsilon subunit of E. coli DNA polymerase III). Nuclear (Pol delta n) and cytosolic (Pol delta c) forms of DNA polymerase delta were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol delta c, Pol delta n had a 20-fold higher exo/pol ratio and allowed 4-5 times higher fidelity of DNA synthesis. The mistake rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.     

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.     

Functional interdependence of DNA polymerizing and 3'-->5' exonucleolytic activities in Pyrococcus furiosus DNA polymerase I

Pyrococcus furiosus DNA polymerase I (Pol BI) belongs to the family B (alpha-like) DNA polymerases and has a strong 3'-->5' exonucleolytic activity, in addition to its DNA polymerizing activity. To understand the relationship between the structure and function of this DNA polymerase, three deletion mutants, Delta1 (DeltaLeu746-Ser775), Delta2 (DeltaLeu717-Ser775) and Delta3 (DeltaHis672-Ser775), and two substituted mutants of Asp405, D405A and D405E, were constructed. These substitutions affected both the DNA polymerizing and the 3'-->5' exonucleolytic activities. The Delta1 mutant protein had DNA polymerizing activity with higher specific activity than that of the wild-type Pol BI, but retained only 10% of the exonucleolytic activity of the wild-type. The other two deletion mutants lost most of both activities. These results suggest that the DNA polymerizing and exonucleolytic activities are closely related to each other in the folded structure of this DNA polymerase, as proposed in the family B DNA polymerases.     

Baculovirus alkaline nuclease possesses a 5'-->3' exonuclease activity and associates with the DNA-binding protein LEF-3

Alkaline nuclease (AN) of the Autographa californica multiple-capsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133) was expressed in recombinant baculovirus as a His(6)-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, AcMNPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with (32)P at the 5' or 3' ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5'-->3' direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 5'-->3' exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Redalpha) and DNA-binding protein (Redbeta) of the Red-mediated homologous recombination system of bacteriophage lambda.     

Replication protein A promotes 5'-->3' end processing during homology-dependent DNA double-strand break repair

Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])-binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3' single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)-mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)-mediated degradation of the 5' ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5'-strand resection of DNA that carries a 3' ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3' ss-DNA for homology-dependent DSB repair.     

Excision of 3' termini by the Trex1 and TREX2 3'-->5' exonucleases. Characterization of the recombinant proteins

The excision of nucleotides from DNA 3' termini is an important step in DNA replication, repair, and recombination pathways to generate correctly base paired termini for subsequent processing. The mammalian TREX1 and TREX2 proteins contain potent 3'-->5' exonucleases capable of functioning in this capacity. To study the activities of these exonucleases we have developed strategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical characterization. The Trex1 and TREX2 proteins are homodimers that exhibit robust 3' excision activities with very similar preferred reaction conditions and preferences for specific DNA substrates. In a steady-state kinetic analysis, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2. The Michaelis constants derived from these data indicate similar apparent kcat values of 22 s(-1) for Trex1 and 16 s(-1) for TREX2 using single-stranded oligonucleotides. The apparent KM values of 19 nm for Trex1 and 190 nm for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2. An exonuclease competition assay was designed using heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2. The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for producing correctly base paired 3' termini.     

Analysis of MRE11's function in the 5'-->3' processing of DNA double-strand breaks

The resection of DNA double-strand breaks (DSBs) into 3' single-strand tails is the initiating step of homology-dependent repair pathways. A key player in this process is the MRE11-RAD50-NBS1 complex, but its contribution to and mechanistic role in resection are not well understood. In this study, we took advantage of the Xenopus egg extract system to address these questions. We found that depletion of MRE11 caused a dramatic inhibition of 5'-resection, even for the first nucleotide at the 5'-end. Depletion of Xenopus CtIP also inhibited 5'-strand resection, but this inhibition could be alleviated by excess MRN. Both MRE11 and CtIP could be bypassed by a DNA that carried a 3'-ss-tail. Finally, using purified proteins, we found that MRN could stimulate both the WRN-DNA2-RPA pathway and the EXO1 pathway of resection. These findings provide important insights into the function of MRE11 in 5'-strand resection.     

Antimutagenic role of autonomous 3'-->5'-exonucleases

Our own and literary data about antimutagenic role of autonomous 3'-->5'-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but often involved in replicative complexes. Intracellular overproduction of AE in bacteria is accompanied with the sharp suppression of mutagenesis, whereas the inactivation of AE in bacteria and higher fungi results in the increase of mutation rates by 2-3 orders of magnitude. The addition of AE in biologically meaningful concentrations to DNA polymerases elevates substantially the accuracy of their work in vitro. In these cases, the reverse mutation rates were measured in the DNA from phage (X174 amber 3, whereas the direct mutation rates--in the DNA from phage M13mp2, both being used as primer-templates for DNA synthesis and then transfected into spheroplasts of Escherichia coli. The accuracy of action of nuclease-free DNA polymerases alpha and beta are shown to raise in the presence of AE by 2-3 orders, the accuracy of moderately processive DNA polymerase I--by 2 orders, the accuracy of highly processive DNA polymerase delta--by 5-10 times, though the latter 2 polymerases display and their own 3'-->5'-exonucleolytic activity. AE, involved in the multienzyme DNA polymerase complexes, augment the accuracy of complexes action by 5-10 times. The model of "external" corrective role of AE in DNA biosynthesis is proposed. Study of 30 objects from all 3 kingdoms of live beings (from archae- and eubacteria to mammalia including human) has shown that AE account, as minimum, from 30 to 90% of the total cellular 3'-->5'-exonucleolytic activity. So AE increase essentially the intracellular ratio of values of 3'-->5'-exonuclease to DNA polymerase activities in the very various representatives from a phylogenetic tree that results always in the augmentation of the accuracy of DNA biosynthesis.     

The dhp1(+) gene, encoding a putative nuclear 5'-->3' exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1⁺ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)⁺ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1⁺ is required for proper chromosome segregation as well as for poly(A)⁺ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1⁺. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.