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Structural and Mechanistic Studies of Nucleic Acid Processing

Research Summary

Marcin Nowotny uses protein crystallography and biochemistry to elucidate the mechanism of proteins involved in nucleic acid processing.

The genetic information stored in deoxyribonucleic acid (DNA) has to be maintained and processed. In my laboratory, we are interested in elucidating the mechanisms of these processes, and to this end we study proteins involved in them using biochemical methods and crystallography. The latter method allows us to determine the positions of all atoms in the protein molecule or, in other words, determine its crystal structure. I am particularly interested in obtaining structures of proteins in complexes with the nucleic acids they act upon. Such structures are the most informative since they provide snapshots of proteins performing their functions.

One particular process I study is DNA repair. Genetic information incurs chemical damage, which has to be repaired in order to maintain the stability of the cell. One type of damage we are interested in occurs while the DNA is copied during cell divisions. Very frequently, ribonucleotides are mistakenly incorporated into the nascent DNA. They are mutagenic and have to be removed from the DNA. The only known enzyme that is capable of this excision is RNase H2. We obtained the crystal structure of this enzyme bound to a fragment of DNA that contained a ribonucleotide. The structure revealed that the enzyme uses two closely spaced metal ions to execute the cleavage of the RNA embedded in the DNA. In a very unique mechanism, the enzyme deforms the junction between RNA and DNA so that the nucleic acid participates in the binding of the metal ions and in this way facilitates its own hydrolysis. Because the deformations are specific to the RNA–DNA junction, the enzyme only cuts at the desired locations.

Human RNase H2 is a complex of three proteins, and mutations in any of them lead to a severe genetic disease called Aicardi-Goutières syndrome (AGS). We determined a crystal structure of human RNase H2 complex that provided insights into the mechanism of this enzyme and also allowed us to map the positions of the mutations from AGS patients. This information allowed us to predict the molecular basis of the defects these mutations cause in the structure and mechanism of human RNase H2.

We also study a very general DNA repair pathway called nucleotide excision repair (NER). Its main feature is the ability to recognize a wide variety of different chemical modifications of the DNA. We solved a crystal structure of bacterial NER protein called UvrA. It functions as the sensor of DNA damage. Our structure demonstrated that UvrA protein does not probe the chemical modification (or damage) itself. Instead, it detects the deformations of the global structure of the DNA double helix induced by the chemical modification. Since many different chemical modifications induce similar deformations of the DNA, UvrA is able to achieve its broad specificity.

I am also interested in reverse transcription. It is an unusual process in which RNA is converted back to DNA. It is a complex multistep chemical reaction catalyzed by a versatile and fascinating enzyme called reverse transcriptase. Reverse transcription is one of the key steps in the replication of retroviruses such as HIV. Their genetic information is encoded in RNA and has to be converted to DNA, which is then incorporated in the genome of the infected cell. We perform structural and biochemical studies of reverse transcriptases to elucidate the mechanism of several critical steps of the reactions they catalyze.

My future plans include the studies of the degradation of the main class of RNA called messenger RNA (mRNA). These molecules are used to synthesize the proteins in the cell. The level of protein production has to be tightly regulated, and one of the main ways to achieve this is to regulate the rate of mRNA production and degradation. The latter starts with the removal of a long stretch of adenosines (or poly(A) tail) attached to each mRNA. Specialized enzymes are responsible for this adenosine removal step, and I plan to study them using crystallography and biochemistry. In humans, these enzymes are composed of two to 10 individual proteins, so they are very complex and challenging objects to study. Our aim is not only to determine the structure and arrangement of their individual components but also to explain how they function and how they are linked to other regulatory processes occurring in the cell.

A Wellcome Trust International Senior Research Fellowship, an EMBO Installation Grant, and an EU FP7 HealthPort grant provided support for these projects.

As of January 17, 2012