Like many other life forms, bacteria too are prone to viral infections. Many bacteria harbor restriction endonuclease enzymes, which help check the infection by selectively cutting and destroying the viral genomic DNA. The perpetual tussle between bacteria and viruses to gain an upper hand has resulted in a battery of restriction enzymes of different complexities. There are the simple nucleases that cut DNA at target sites – two copies of the nuclease come together, each cutting a strand to slice the double-stranded DNA. And there are motor-driven nucleases that are massive in size and use chemical energy to cut DNA only upon collision with another such nuclease and away from the target sites. A collaborative study between a team of scientists at IISER Pune and the University of Bristol, UK, of these energy-driven nucleases, published this week in Nature Chemical Biology, reveals a new mechanism of DNA break formation involving a compound damage caused by DNA shredding rather than slicing.
Dr. Saikrishnan Kayarat’s team at IISER Pune has solved the first atomic resolution x-ray crystal structure of a motor-driven restriction endonuclease bound to DNA. They found that contrary to the prevalently understood mode of action, the nuclease domain of this class of enzyme is positioned such that when two enzymes collide on a DNA, the nuclease domains are distant from each other. This structure also happens to be of one of the largest single-polypeptide chain bound to nucleic acid determined to date.
Guided by the structure, Prof. Mark Szczelkun and colleagues at the University of Bristol used single-molecule biophysical approaches, to find that the nucleases use the energy derived from the cellular fuel, ATP, to run along the DNA. Using biochemical approaches they found that upon collision, the distantly spaced nucleases make multiple nicks on the individual strands, thus shredding the double stranded DNA. This is in contrast to a clean-cut slicing brought about by the action of an enzyme with an apposed pair of nuclease domains. Unlike a sliced-DNA, a shredded DNA cannot be easily repaired.
The paper titled “Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes” and authored by Mahesh K. Chand, Neha Nirwan, Fiona M Diffin, Kara van Aelst, Manasi Kulkarni, Christian Pernstich, Mark D. Szczelkun and Kayarat Saikrishnan has appeared as an advance online publication of Nature Chemical Biology.
This work received funding from Wellcome Trust-DBT India Alliance; Wellcome Trust, UK; DBT India; and CSIR India.