Mechanism of DNA Recognition by the Restriction Enzyme EcoRV

Abstract

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV-cognate DNA crystallographic complex, a sharp kink of 50 degrees has been found at the center base-pair step (TA). Here, we examine the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme by performing all-atom molecular dynamics simulations of EcoRV unbound and interacting with three DNA sequences: the cognate sequence, GATATC (TA); the non-cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GA(CH(3))TATC (TA-CH(3)). In the unbound EcoRV, the cleft between the two C-terminal subdomains is found to be open. Binding to AT narrows the cleft and forms a partially bound state. However, the intrinsic bending propensity of AT is insufficient to allow tight binding. In contrast, the cognate TA sequence is easier to bend, allowing specific, high-occupancy hydrogen bonds to form in the complex. The absence of cleavage for this methylated sequence is found to arise from the loss of specific hydrogen bonds between the first adenine of the recognition sequence and Asn185. On the basis of the results, we suggest a three-step recognition mechanism. In the first step, EcoRV, in an open conformation, binds to the DNA at a random sequence and slides along it. In the second step, when the two outer base pairs, GAxxTC, are recognized, the R loops of the protein become more ordered, forming strong hydrogen-bonding interactions, resulting in a partially bound EcoRV-DNA complex. In the third step, the flexibility of the center base pair is probed, and in the case of the full cognate sequence the DNA bends, the complex strengthens and the protein and DNA interact more closely, allowing cleavage.

The Energetic Contribution of Induced Electrostatic Asymmetry to DNA Bending by a Site-Specific Protein

“…MD simulations show that the harmonic force constant for roll is lowest at a T-A step 79 , in accord with the rank order of roll dispersions based on crystallographic survey 23 . A recent computational study 80 shows that although the most probable bend angle at the protein-free GATATC site is close to zero (i.e., straight DNA), the fraction of the population assuming bend angles of 10° to 30° is substantially greater than for a GAATTC site lacking the T-A step.…”

Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease

“…MD simulations of cognate‐DNA–EcoRV complexed with only (1TA) are compared to results of simulations published previously of cognate‐DNA–EcoRV (2TA) and non‐cognate‐DNA–EcoRV (AT), both complexed with two ions ( and ) [19]. The non‐cognate DNA sequence GAATTC is known not to be cleaved by EcoRV [21].…”

“…Computational work has demonstrated the usefulness of MD simulation in understanding the interplay between the structural properties of nucleic acids and their interactions with the protein partners [18–20]. Here, MD simulations are performed to examine the role of Metal B in the structure of the EcoRV–DNA complex.…”

The Effect of a G:T Mispair on the Dynamics of DNA

“…Molecular simulations have proven to be a powerful tool for obtaining information on structure and dynamics at the atomic level which is not directly accessible to experiments, and have been used successfully to analyse conformational changes in proteins and DNA [13]–[27].…”