CH391L/S2013 Alesha Stewart Mar 20 2013

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(Background & Introduction)
(Methods)
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==Methods==
==Methods==
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===DNA and MutM Synthesis===
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Mutant MutM proteins were synthesized from B. stearothermophilus MutM gene with point mutations and megaprimer mutagenesis. The DNA oligomers with and without lesions were synthesized using a DNA synthesizer. Phosphoramidate- cystamine backbone modifications were also made for DXL with the cysteine-engineered MutM.
 +
===Crosslink formation===
 +
Different length carbon linkers and phosphate positions on the DNA backbone were investigated for DXL of MutM. Two, three, and four carbon chain thiols were tethered to the phosphates to serve as the linker to attach the cys-engineered protein during incubation (2:1 protein to DNA concentration). Non-reducing SDS-PAGE confirmed the crosslinking quality fig S1, and the product was purified on a monoQ column and concentrated to 250 μM.
 +
 +
===Crystallization===
 +
X-ray crystallography was used to determine the crystal structures of the protein-DNA complexes. X-rays were generated using a synchrotron.
 +
 +
OxoG containing complexes were crystallized using the 1:1 hanging drop vapor diffusion method (13−18% (w/v) PEG 8K, 100 mM sodium cacodylate, pH 7.5, 50 mM Mg(CH3COO)2 and 0.1 % βME reservoir solution), cryoprotected with glycerol, and preserved in liquid nitrogen for data collection. There was initial model for refinement from the isomorphous structure of the LRC of MutM. Then the model was built with continuous rounds of rigid body refinement, energy minimization, simulated annealing and grouped B-factor refinement.
 +
 +
Crosslinked, undamaged complexes were formed using the 1:1 hanging drop vapor diffusion method at 4°C (12-18% PEG 8K, 100mM sodium cacodylate pH 7.0 and 0-5% glycerol reservoir solution). These crystals were also cryoprotected with glycerol and frozen in liquid nitrogen for data collection. Crystal structures were solved with molecular replacement, which requires a model of the structure in advance to serve as the initial search model to conduct the translation and cross rotation search. The models were built as previously stated.
==Results==
==Results==

Revision as of 11:08, 20 March 2013

User:Alesha Stewart

Structure of a DNA glycosylase searching for lesions- Banerjee A, Santos WL, Verdine GL


Contents

Background & Introduction

DNA glycosylases are proteins responsible for DNA damage recognition and repair [4]. These enzymes search entire genomes for single nucleobase lesions to initiate base excision repair. The ability of DNA glycosylase to seek and find damaged bases is a wonder of science, especially since there are only minor differences between impaired and unimpaired bases. Thermo energy fuels this efficient process, as no biochemical forms of energy are expended. Details concerning the function and execution of DNA glycosylase are still under investigation, but the article “The Structure of a DNA Glycosylase Searching for Lesions” intends to shed light on the subject [1].

In this study, the authors utilized a bacterial DNA glycosylase (Bacillus stearothermophilus MutM) that specifically associates with 8-oxoguanine (oxoG). OxoG is a form of DNA damage from reactive oxygen species, where guanine is oxidized. As a result, the lesion can cause mismatched base pairing or mutations leading to genome instability and cancer if the damage goes unrepaired [2]. There is an oxoG resistance pathway in bacteria to protect against such damage (the “GO” system). MutM is the component of the system responsible for excising oxoG:C base pairs [3].

When MutM binds to an oxoG DNA base it represents the lesion recognition complex (LRC), which was used to investigate the recognition and repair method Fig 1A. Images of the LRC indicated that MutM physically flips the oxidized nucleobase out from the DNA helix to position it into its active site Fig 1A. Extrahelical base excision is now believed to be a universal technique used for all DNA glycosylases [1].

The mechanism of how extrahelical base excision is executed, however, is still undetermined. There are three proposals previously considered to explain how DNA glycosylases search, find, and repair damaged nucleobases- 1) It actively extrudes every base from the DNA; 2) It detects damaged bases that were spontaneously extruded from the DNA; or 3) It identifies lesions and activates the extrusion from the DNA. The researchers focused on the latter option, intrahelical lesion recognition, as it is the most kinetically favorable [1].

Methods

DNA and MutM Synthesis

Mutant MutM proteins were synthesized from B. stearothermophilus MutM gene with point mutations and megaprimer mutagenesis. The DNA oligomers with and without lesions were synthesized using a DNA synthesizer. Phosphoramidate- cystamine backbone modifications were also made for DXL with the cysteine-engineered MutM.

Crosslink formation

Different length carbon linkers and phosphate positions on the DNA backbone were investigated for DXL of MutM. Two, three, and four carbon chain thiols were tethered to the phosphates to serve as the linker to attach the cys-engineered protein during incubation (2:1 protein to DNA concentration). Non-reducing SDS-PAGE confirmed the crosslinking quality fig S1, and the product was purified on a monoQ column and concentrated to 250 μM.

Crystallization

X-ray crystallography was used to determine the crystal structures of the protein-DNA complexes. X-rays were generated using a synchrotron.

OxoG containing complexes were crystallized using the 1:1 hanging drop vapor diffusion method (13−18% (w/v) PEG 8K, 100 mM sodium cacodylate, pH 7.5, 50 mM Mg(CH3COO)2 and 0.1 % βME reservoir solution), cryoprotected with glycerol, and preserved in liquid nitrogen for data collection. There was initial model for refinement from the isomorphous structure of the LRC of MutM. Then the model was built with continuous rounds of rigid body refinement, energy minimization, simulated annealing and grouped B-factor refinement.

Crosslinked, undamaged complexes were formed using the 1:1 hanging drop vapor diffusion method at 4°C (12-18% PEG 8K, 100mM sodium cacodylate pH 7.0 and 0-5% glycerol reservoir solution). These crystals were also cryoprotected with glycerol and frozen in liquid nitrogen for data collection. Crystal structures were solved with molecular replacement, which requires a model of the structure in advance to serve as the initial search model to conduct the translation and cross rotation search. The models were built as previously stated.

Results

Conclusion & Significance

References

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