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Demethylation initiated by ROS1 glycosylase involves random sliding along DNA.

Ponferrada-Marín MI, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2012)

Bottom Line: REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway.We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein.Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba/IMIBIC, 14071 Córdoba, Spain.

ABSTRACT
Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.

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A model for 5-meC and T recognition by ROS1. The model is based on that proposed in (40) for DNA glycosylase Fpg, and postulates two recognition modes. In the ‘run-on mode’, the enzyme binds non-specifically and approaches its target by sliding along the DNA. In the ‘jump-on’ mode, the protein binds in the immediate vicinity of the target. ROS1 would form productive catalytic complexes in both modes with 5-meC:G pairs (A), but only in the ‘jump-on’ mode with T:G mispairs (B). Productive encounters are indicated by bold arrows, and protein motion is represented by thin arrows. See text for details.
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gks894-F6: A model for 5-meC and T recognition by ROS1. The model is based on that proposed in (40) for DNA glycosylase Fpg, and postulates two recognition modes. In the ‘run-on mode’, the enzyme binds non-specifically and approaches its target by sliding along the DNA. In the ‘jump-on’ mode, the protein binds in the immediate vicinity of the target. ROS1 would form productive catalytic complexes in both modes with 5-meC:G pairs (A), but only in the ‘jump-on’ mode with T:G mispairs (B). Productive encounters are indicated by bold arrows, and protein motion is represented by thin arrows. See text for details.

Mentions: A plausible model suggesting two different modes of lesion finding has been previously proposed for the DNA glycosylase Fpg, which excises 8-oxo-7,8-dihydroguanine and less efficiently 5,6-dihydrouracil (40). We have adapted this model to explain 5-meC and T recognition by ROS1 (Figure 6). The model postulates two modes of target location. In the ‘run-on’ mode, the enzyme approaches the target while sliding along DNA, whereas in the ‘jump-on’ mode, it binds in the immediate vicinity of the target. We propose that ROS1 forms productive catalytic complexes in both modes with 5-meC:G pairs, but only in the latter mode with T:G mispairs (Figure 6). Similarly to other DNA glycosylases, it is likely that during sliding, ROS1 forms a transient ‘interrogation complex’ that would extrude bases for inspection (41). We propose that such ‘interrogation complex’ would convert into a catalytically productive ‘excision complex’ on encountering 5-meC, but not T. This mechanism might limit the risk that an extruded correctly paired T attains the pre-catalytic state of the base extrusion pathway. Thus, the structural and mechanistic features of ROS1 might have evolved to simultaneously optimize excision of 5-meC and prevent aberrant activity on an overwhelmingly abundant suboptimal target.Figure 6.


Demethylation initiated by ROS1 glycosylase involves random sliding along DNA.

Ponferrada-Marín MI, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2012)

A model for 5-meC and T recognition by ROS1. The model is based on that proposed in (40) for DNA glycosylase Fpg, and postulates two recognition modes. In the ‘run-on mode’, the enzyme binds non-specifically and approaches its target by sliding along the DNA. In the ‘jump-on’ mode, the protein binds in the immediate vicinity of the target. ROS1 would form productive catalytic complexes in both modes with 5-meC:G pairs (A), but only in the ‘jump-on’ mode with T:G mispairs (B). Productive encounters are indicated by bold arrows, and protein motion is represented by thin arrows. See text for details.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526269&req=5

gks894-F6: A model for 5-meC and T recognition by ROS1. The model is based on that proposed in (40) for DNA glycosylase Fpg, and postulates two recognition modes. In the ‘run-on mode’, the enzyme binds non-specifically and approaches its target by sliding along the DNA. In the ‘jump-on’ mode, the protein binds in the immediate vicinity of the target. ROS1 would form productive catalytic complexes in both modes with 5-meC:G pairs (A), but only in the ‘jump-on’ mode with T:G mispairs (B). Productive encounters are indicated by bold arrows, and protein motion is represented by thin arrows. See text for details.
Mentions: A plausible model suggesting two different modes of lesion finding has been previously proposed for the DNA glycosylase Fpg, which excises 8-oxo-7,8-dihydroguanine and less efficiently 5,6-dihydrouracil (40). We have adapted this model to explain 5-meC and T recognition by ROS1 (Figure 6). The model postulates two modes of target location. In the ‘run-on’ mode, the enzyme approaches the target while sliding along DNA, whereas in the ‘jump-on’ mode, it binds in the immediate vicinity of the target. We propose that ROS1 forms productive catalytic complexes in both modes with 5-meC:G pairs, but only in the latter mode with T:G mispairs (Figure 6). Similarly to other DNA glycosylases, it is likely that during sliding, ROS1 forms a transient ‘interrogation complex’ that would extrude bases for inspection (41). We propose that such ‘interrogation complex’ would convert into a catalytically productive ‘excision complex’ on encountering 5-meC, but not T. This mechanism might limit the risk that an extruded correctly paired T attains the pre-catalytic state of the base extrusion pathway. Thus, the structural and mechanistic features of ROS1 might have evolved to simultaneously optimize excision of 5-meC and prevent aberrant activity on an overwhelmingly abundant suboptimal target.Figure 6.

Bottom Line: REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway.We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein.Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba/IMIBIC, 14071 Córdoba, Spain.

ABSTRACT
Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.

Show MeSH
Related in: MedlinePlus