Next generation gene synthesis: From microarrays to genomes.

Similar to the incredible advances in DNA sequencing, the de novo synthesis of DNA is subject to innovations and fast progress in terms of synthesis speed and cost. We will discuss novel techniques that are expected to enable high-throughput synthesis of oligonucleotides on microarrays and the subsequent assembly into longer fragments, up to whole genomes. Especially, the inherent disadvantages of microarray-derived oligonucleotide pools for gene synthesis will be discussed in detail, and also the different approaches to still render these oligonucleotides useful for gene assembly. These so-called next-generation techniques will lead to a significant cost reduction of gene synthesis and to the possibility of much larger projects, such as whole genome synthesis.

Rescuing DNA repair activity by rewiring the H-atom transfer pathway in the radical SAM enzyme, spore photoproduct lyase.

The radical SAM enzyme, spore photoproduct lyase, requires an H-atom transfer (HAT) pathway to catalyze DNA repair. By rational engineering, we demonstrate that it is possible to rewire its HAT pathway, a first step toward the development of novel catalysts based on the radical SAM enzyme scaffold.

Formation and Direct Repair of UV-induced Dimeric DNA Pyrimidine Lesions.

Direct repair of UV-induced DNA lesions represents an elegant method for many organisms to deal with these highly mutagenic and cytotoxic compounds. Although the participating proteins are structurally well investigated, the exact repair mechanism of the photolyase enzymes remains a vivid subject of current research. In this review, we summarize and highlight the recent contributions to this exciting field.

Strand-specific recognition of DNA damages by XPD provides insights into nucleotide excision repair substrate versatility.

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

The radical SAM enzyme spore photoproduct lyase employs a tyrosyl radical for DNA repair.

The spore photoproduct lyase is a radical SAM enzyme, which repairs 5-(α-thyminyl)-5,6-dihydrothymidine. Here we show that the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction.

Structural insights into recognition and repair of UV-DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme.

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase) that repairs a specific UV-induced DNA lesion, the spore photoproduct (SP), through an unprecedented radical-based mechanism. Unlike DNA photolyases, SP lyase belongs to the emerging superfamily of radical S-adenosyl-l-methionine (SAM) enzymes and uses a [4Fe-4S](1+) cluster and SAM to initiate the repair reaction. We report here the first crystal structure of this enigmatic enzyme in complex with its [4Fe-4S] cluster and its SAM cofactor, in the absence and presence of a DNA lesion, the dinucleoside SP. The high resolution structures provide fundamental insights into the active site, the DNA lesion recognition and binding which involve a ß-hairpin structure. We show that SAM and a conserved cysteine residue are perfectly positioned in the active site for hydrogen atom abstraction from the dihydrothymine residue of the lesion and donation to the α-thyminyl radical moiety, respectively. Based on structural and biochemical characterizations of mutant proteins, we substantiate the role of this cysteine in the enzymatic mechanism. Our structure reveals how SP lyase combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place.

ONIOM approach for non-adiabatic on-the-fly molecular dynamics demonstrated for the backbone controlled Dewar valence isomerization.

Non-adiabatic on-the-fly molecular dynamics (NA-O-MD) simulations require the electronic wavefunction, energy gradients, and derivative coupling vectors in every timestep. Thus, they are commonly restricted to the excited state dynamics of molecules with up to ≈20 atoms. We discuss an approximation that combines the ONIOM(QM:QM) method with NA-O-MD simulations to allow calculations for larger molecules. As a proof of principle we present the excited state dynamics of a (6-4)-lesion containing dinucleotide (63 atoms), and especially the importance to include the confinement effects of the DNA backbone. The method is able to include electron correlation on a high level of theory and offers an attractive alternative to QM:MM approaches for moderate sized systems with unknown force fields.

Mechanism of UV-induced formation of Dewar lesions in DNA.

The importance of a backbone: The mechanism of formation of Dewar lesions has been investigated by using femtosecond IR spectroscopy and ab initio calculations of the exited state. The 4π electrocyclization is rather slow, occurs with an unusual high quantum yield, and--surprisingly--is controlled by the phosphate backbone.

Crystal structures and repair studies reveal the identity and the base-pairing properties of the UV-induced spore photoproduct DNA lesion.

UV light is one of the major causes of DNA damage. In spore DNA, due to an unusual packing of the genetic material, a special spore photoproduct lesion (SP lesion) is formed, which is repaired by the enzyme spore photoproduct lyase (Spl), a radical S-adenosylmethionine (SAM) enzyme. We report here the synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone. The oligonucleotides were used for repair studies and they were cocrystallized with a polymerase enzyme as a template to clarify the configuration of the SP lesion and to provide information about the base-pairing properties of the lesion. The structural analysis together with repair studies allowed us to clarify the identity of the preferentially repaired lesion diastereoisomer.

Investigation of excess-electron transfer in DNA double-duplex systems allows estimation of absolute excess-electron transfer and CPD cleavage rates.

To investigate the parameters and rates that determine excess-electron transfer processes in DNA duplexes, we developed a DNA double-duplex system containing a reduced and deprotonated flavin donor at the junction of two duplexes with either the same or different electron acceptors in the individual duplex substructures. This model system allows us to bring the two electron acceptors in the duplex substructures into direct competition for injected electrons and this enables us to decipher how the kind of acceptor influences the transfer data. Measurements with the electron acceptors 8-bromo-dA (BrdA), 8-bromo-dG (BrdG), 5-bromo-dU (BrdU), and a cyclobutane pyrimidine dimer, which is a UV-induced DNA lesion, allowed us to obtain directly the maximum overall reaction rates of these acceptors and especially of the T=T dimer with the injected electrons in the duplex. In line with previous observations, we detected that the overall dimer cleavage rate is about one order of magnitude slower than the debromination of BrdU. Furthermore, we present a more detailed explanation of why sequence dependence cannot be observed when a T=T dimer is used as the acceptor and we estimate the absolute excess-electron hopping rates.

Chemical investigation of light induced DNA bipyrimidine damage and repair.

In all organisms, genetic information is stored in DNA and RNA. Both of these macromolecules are damaged by many exogenous and endogenous events, with UV irradiation being one of the major sources of damage. The major photolesions formed are the cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidone-(6-4)-photoproducts, Dewar valence isomers and, for dehydrated spore DNA, 5-(α-thyminyl)-5,6-dihydrothymine (SP). In order to be able to investigate how nature's repair and tolerance mechanisms protect the integrity of genetic information, oligonucleotides containing sequence and site-specific UV lesions are essential. This tutorial review provides an overview of synthetic procedures by which these oligonucleotides can be generated, either through phosphoramidite chemistry or direct irradiation of DNA. Moreover, a brief summary on their usage in analysing repair and tolerance processes as well as their biological effects is provided.

DNA (6-4) photolyases reduce Dewar isomers for isomerization into (6-4) lesions.

Repair of the Dewar valence isomers by (6-4) photolyases proceeds via an enzyme catalyzed ring-opening reaction of the Dewar lesion to the (6-4) photoproduct.