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Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues.

Parrilla-Doblas JT, Ponferrada-Marín MI, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2013)

Bottom Line: Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs.While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA.Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba/Maimónides Institute for Research in Biomedicine of Córdoba (IMIBIC)/Reina Sofía University Hospital, 14071 Córdoba, Spain.

ABSTRACT
Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.

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Stable binding of Q607A variant to a DNA substrate with blocked ends. Purified WT ROS1 (upper panel, 130 nM) or Q607A mutant variant (lower panel, 130 nM) were incubated at 25°C with 100 nM of fluorescein-labeled substrates S (lanes 2–6) or SL1-2 (lanes 7–11), both containing a single 5-meC:G pair, and the reactions were monitored for 60 min. After nondenaturing gel electrophoresis, the gel was scanned to detect fluorescein-labeled DNA. Protein–DNA complexes were identified by their retarded mobility compared with that of free DNA, as indicated. Graphs on the left show the percentage of protein–DNA complexes at different incubation times. Values are mean ± SE (error bars) from three independent experiments.
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gkt625-F7: Stable binding of Q607A variant to a DNA substrate with blocked ends. Purified WT ROS1 (upper panel, 130 nM) or Q607A mutant variant (lower panel, 130 nM) were incubated at 25°C with 100 nM of fluorescein-labeled substrates S (lanes 2–6) or SL1-2 (lanes 7–11), both containing a single 5-meC:G pair, and the reactions were monitored for 60 min. After nondenaturing gel electrophoresis, the gel was scanned to detect fluorescein-labeled DNA. Protein–DNA complexes were identified by their retarded mobility compared with that of free DNA, as indicated. Graphs on the left show the percentage of protein–DNA complexes at different incubation times. Values are mean ± SE (error bars) from three independent experiments.

Mentions: We have recently reported that ROS1 performs sliding on DNA while searching for its target base (22). Therefore, we reasoned that if Q607 plays any role in DNA interrogation, the absence of this residue in the mutant protein should have an effect on DNA sliding. To examine this possibility, we compared the diffusive behavior of WT ROS1 and Q607A on a substrate containing tetraloop obstacles along the DNA surface (Figure 6). We preincubated WT ROS1 or Q607A with labeled substrates S or SL1-2 and then added increasing concentrations of unlabeled S competitor to promote dissociation. Consistent with our previously reported observations (22), we found that WT ROS1 dissociates from substrate S when chased by the competitor, but remains bound to substrate SL1-2 even at high competitor concentrations. As expected, Q607A was unable to bind substrate S. However, the mutant protein was able to form a stable complex with substrate SL1-2, resisting competition with increasing concentrations of unlabeled S (Figure 6). By performing DNA binding measurements at different time points in the absence of competitor, we detected stable complexes of WT ROS1 with both S and SL1-2, whereas Q607A only bound stably to SL1-2 (Figure 7). Furthermore, we found that unblocking one of the substrate ends greatly reduced the capacity of Q607A to form a stable complex with DNA (Supplementary Figure S5). We therefore conclude that the Q607A variant is unable to form a stable complex with a DNA substrate containing free ends, but remains bound to a molecule whose ends are obstructed with tetraloop blocks. Importantly, we found that the stable binding of Q607A to substrate SL1-2 did not have any positive effect on the reduced catalytic activity of the mutant protein (Supplementary Figure S6), thus corroborating the essential role of Q607 for base excision. Altogether, these results indicate that Q607 has an inhibitory effect on ROS1 sliding along DNA, and suggest that the enzyme extrudes unmethylated bases for interrogation by inserting this residue into the base stack.Figure 6.


Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues.

Parrilla-Doblas JT, Ponferrada-Marín MI, Roldán-Arjona T, Ariza RR - Nucleic Acids Res. (2013)

Stable binding of Q607A variant to a DNA substrate with blocked ends. Purified WT ROS1 (upper panel, 130 nM) or Q607A mutant variant (lower panel, 130 nM) were incubated at 25°C with 100 nM of fluorescein-labeled substrates S (lanes 2–6) or SL1-2 (lanes 7–11), both containing a single 5-meC:G pair, and the reactions were monitored for 60 min. After nondenaturing gel electrophoresis, the gel was scanned to detect fluorescein-labeled DNA. Protein–DNA complexes were identified by their retarded mobility compared with that of free DNA, as indicated. Graphs on the left show the percentage of protein–DNA complexes at different incubation times. Values are mean ± SE (error bars) from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794587&req=5

gkt625-F7: Stable binding of Q607A variant to a DNA substrate with blocked ends. Purified WT ROS1 (upper panel, 130 nM) or Q607A mutant variant (lower panel, 130 nM) were incubated at 25°C with 100 nM of fluorescein-labeled substrates S (lanes 2–6) or SL1-2 (lanes 7–11), both containing a single 5-meC:G pair, and the reactions were monitored for 60 min. After nondenaturing gel electrophoresis, the gel was scanned to detect fluorescein-labeled DNA. Protein–DNA complexes were identified by their retarded mobility compared with that of free DNA, as indicated. Graphs on the left show the percentage of protein–DNA complexes at different incubation times. Values are mean ± SE (error bars) from three independent experiments.
Mentions: We have recently reported that ROS1 performs sliding on DNA while searching for its target base (22). Therefore, we reasoned that if Q607 plays any role in DNA interrogation, the absence of this residue in the mutant protein should have an effect on DNA sliding. To examine this possibility, we compared the diffusive behavior of WT ROS1 and Q607A on a substrate containing tetraloop obstacles along the DNA surface (Figure 6). We preincubated WT ROS1 or Q607A with labeled substrates S or SL1-2 and then added increasing concentrations of unlabeled S competitor to promote dissociation. Consistent with our previously reported observations (22), we found that WT ROS1 dissociates from substrate S when chased by the competitor, but remains bound to substrate SL1-2 even at high competitor concentrations. As expected, Q607A was unable to bind substrate S. However, the mutant protein was able to form a stable complex with substrate SL1-2, resisting competition with increasing concentrations of unlabeled S (Figure 6). By performing DNA binding measurements at different time points in the absence of competitor, we detected stable complexes of WT ROS1 with both S and SL1-2, whereas Q607A only bound stably to SL1-2 (Figure 7). Furthermore, we found that unblocking one of the substrate ends greatly reduced the capacity of Q607A to form a stable complex with DNA (Supplementary Figure S5). We therefore conclude that the Q607A variant is unable to form a stable complex with a DNA substrate containing free ends, but remains bound to a molecule whose ends are obstructed with tetraloop blocks. Importantly, we found that the stable binding of Q607A to substrate SL1-2 did not have any positive effect on the reduced catalytic activity of the mutant protein (Supplementary Figure S6), thus corroborating the essential role of Q607 for base excision. Altogether, these results indicate that Q607 has an inhibitory effect on ROS1 sliding along DNA, and suggest that the enzyme extrudes unmethylated bases for interrogation by inserting this residue into the base stack.Figure 6.

Bottom Line: Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs.While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA.Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Córdoba/Maimónides Institute for Research in Biomedicine of Córdoba (IMIBIC)/Reina Sofía University Hospital, 14071 Córdoba, Spain.

ABSTRACT
Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.

Show MeSH
Related in: MedlinePlus