RESUMEN
Replicate it: Structures of KOD and 9°N DNA polymerases, two enzymes that are widely used to replicate DNA with highly modified nucleotides, were solved at high resolution in complex with primer/template duplex. The data elucidate substrate interaction of the two enzymes and pave the way for further optimisation of the enzymes and substrates.
Asunto(s)
Cartilla de ADN/química , ADN Polimerasa Dirigida por ADN/química , Sitios de Unión , Cartilla de ADN/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Estructura Terciaria de Proteína , Thermococcus/enzimologíaRESUMEN
The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2'-deoxynucleoside-5'-O-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications.
Asunto(s)
ADN Polimerasa I/química , Nucleósidos de Purina/química , Nucleósidos de Pirimidina/química , Thermus/enzimología , Cristalografía por Rayos X , ADN Polimerasa I/metabolismo , Modelos Moleculares , Polifosfatos/química , Polifosfatos/metabolismo , Unión Proteica , Conformación Proteica , Nucleósidos de Purina/metabolismo , Purinas/química , Purinas/metabolismo , Nucleósidos de Pirimidina/metabolismo , Thermus/químicaRESUMEN
The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase ß when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase ß was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase ß and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase ß variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.
