Final Report Summary - NERCOMP (Structural studies of Nucleotide Excision Repair complexes)
DNA (deoxyribonucleic acid) is a very long molecule present in living cells that encodes a blueprint for each organism. This blueprint is termed genetic information. DNA has two strands encoding mirror images of the same information. It constantly undergoes chemical damage, either spontaneously or as a result of various chemical and physical factors, for example carcinogens, ultraviolet light or radiation. Each organism possesses effective mechanisms to repair those DNA lesions. This repair is very important, because DNA damage usually leads to cell death and, if the cell survives, to typos in the genetic information (mutations) that in longer term lead to cancer in higher organisms.
One of the most important DNA repair pathways is Nucleotide Excision Repair (NER). Its main feature is the ability to detect and repair a wide variety of unrelated chemical modifications. Its first step is the detection of DNA alteration. The presence of the damage is verified and the modified fragment of the damaged DNA strand is cut out and replaced by a fresh piece of DNA. In 2015 the characterization of this pathway was recognized with a Nobel Prize in Chemistry awarded to Aziz Sancar.
In this project we studied NER in bacteria and higher organisms (yeast and human) to understand its selected important elements at the level of single molecules. For the bacterial system we focused on the damage verification step which is performed by an enzyme termed UvrB. The approximate location of the damage is first identified by a protein termed UvrA which detects the deformations of the DNA molecule induced by DNA modification. However, the mechanism of the damage verification by UvrB was unknown. We determined the architecture (structure) of an assembly of UvrA and UvrB on damaged DNA. We found that UvrA brings two UvrB molecules to the DNA. UvrB has the ability to move along the DNA molecule and each UvrB recruited by UvrA travels on a different DNA strand. The one that travels on the damaged strand will be blocked at the exact site of the chemical modification and will bring the third protein – UvrC – which will execute precise cuts in the damaged strand of the DNA. This is how the initial approximate localization of the damage is turned into precise cutting of the DNA to perform the repair. Our goal now is to understand this third step in the pathway and produce a complete description of this process - a type of a movie showing all steps of this DNA repair pathway in bacteria.
The eukaryotic NER uses the same general principles but the proteins involved are different. They are more numerous and more complex. We studied one of the two enzymes that cut the damaged DNA. It is termed Rad2 in yeast and XPG in humans. This protein is important - its defects (mutations) lead to severe human diseases, with high sensitivity to sunlight (UV light), very high predisposition to cancer and premature aging. These are the highlights of defective DNA repair in higher organisms. We determined the three-dimensional structure of Rad2/XPG molecule at the level of individual atoms. Based on this structure we elucidated the mechanism of action of this enzyme – how it finds the proper location to perform the DNA cut and how it binds the special DNA structure which is formed in the process of repair. Moreover, we identified the exact location of the defects (mutations) observed in patients and we explained how those defects influence the structure and activity of this enzyme. It will now be very interesting to see how this enzyme works together with other elements of the eukaryotic NER. The structure also offers possibilities of modulating XPG action for example to sensitize cancer cells to DNA damaging agents, some of which are used as anti-cancer drugs.
One of the most important DNA repair pathways is Nucleotide Excision Repair (NER). Its main feature is the ability to detect and repair a wide variety of unrelated chemical modifications. Its first step is the detection of DNA alteration. The presence of the damage is verified and the modified fragment of the damaged DNA strand is cut out and replaced by a fresh piece of DNA. In 2015 the characterization of this pathway was recognized with a Nobel Prize in Chemistry awarded to Aziz Sancar.
In this project we studied NER in bacteria and higher organisms (yeast and human) to understand its selected important elements at the level of single molecules. For the bacterial system we focused on the damage verification step which is performed by an enzyme termed UvrB. The approximate location of the damage is first identified by a protein termed UvrA which detects the deformations of the DNA molecule induced by DNA modification. However, the mechanism of the damage verification by UvrB was unknown. We determined the architecture (structure) of an assembly of UvrA and UvrB on damaged DNA. We found that UvrA brings two UvrB molecules to the DNA. UvrB has the ability to move along the DNA molecule and each UvrB recruited by UvrA travels on a different DNA strand. The one that travels on the damaged strand will be blocked at the exact site of the chemical modification and will bring the third protein – UvrC – which will execute precise cuts in the damaged strand of the DNA. This is how the initial approximate localization of the damage is turned into precise cutting of the DNA to perform the repair. Our goal now is to understand this third step in the pathway and produce a complete description of this process - a type of a movie showing all steps of this DNA repair pathway in bacteria.
The eukaryotic NER uses the same general principles but the proteins involved are different. They are more numerous and more complex. We studied one of the two enzymes that cut the damaged DNA. It is termed Rad2 in yeast and XPG in humans. This protein is important - its defects (mutations) lead to severe human diseases, with high sensitivity to sunlight (UV light), very high predisposition to cancer and premature aging. These are the highlights of defective DNA repair in higher organisms. We determined the three-dimensional structure of Rad2/XPG molecule at the level of individual atoms. Based on this structure we elucidated the mechanism of action of this enzyme – how it finds the proper location to perform the DNA cut and how it binds the special DNA structure which is formed in the process of repair. Moreover, we identified the exact location of the defects (mutations) observed in patients and we explained how those defects influence the structure and activity of this enzyme. It will now be very interesting to see how this enzyme works together with other elements of the eukaryotic NER. The structure also offers possibilities of modulating XPG action for example to sensitize cancer cells to DNA damaging agents, some of which are used as anti-cancer drugs.