Many type II restriction enzymes have been shown to be able to cut ssDNA.

If single-stranded binding proteins are bound to single-stranded DNA (ssDNA), does this prevent restriction enzymes which cut ssDNA from scanning the ssDNA and interacting with the target cut site?

The NEB website says that using SSB's improve restriction enzyme digestion, so it doesn't seem to be the case that SSBs inhibit target site recognition. But it is unclear to me how a restriction enzyme could recognize the target sequence unless it can also displace SSB's while scanning.

edit: specified REs that cut ssDNA

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    $\begingroup$ I'm curious as to why you are trying to add SSBs to ssDNA endonuclease digestions, is this some in house protocol you are trying? $\endgroup$ Mar 18, 2021 at 1:05
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    $\begingroup$ I was thinking about how ssDNA endonucleases would work in vivo, where there are other proteins like SSB that could potentially interact with the ssDNA and interfere with cutting. $\endgroup$
    – etowah
    Mar 18, 2021 at 21:00
  • $\begingroup$ Please update your question to indicate why you think type II restriction enzymes (that cut double-stranded DNA) would need to displace a single-stranded DNA binding protein. $\endgroup$
    – tyersome
    Mar 24, 2021 at 18:30
  • $\begingroup$ I was thinking about type II restriction enzymes that were capable of cutting ssDNA. $\endgroup$
    – etowah
    Apr 2, 2021 at 4:37
  • $\begingroup$ Do you have some reason to think that this is a normal activity of type II REs? If so, please edit your post to include a summary of that evidence with supporting references. In the absence of that evidence your question doesn't appear to make sense. $\endgroup$
    – tyersome
    Apr 3, 2021 at 22:10

1 Answer 1


This is an interesting point. In the case of typical restriction enzyme digestion, it is double stranded DNA that is being digested. So in this case the SSBs are presumably binding to the sticky ends after digestion, so there is no competition with the restriction enzyme.

If you are interested in a specific case where you are digesting ssDNA, and have decided to add in single strand binding proteins, the binding strength (rate constants) of the SSB and restriction enzyme would influence how much the enzyme can displace the binding protein.

Here's a paragraph from the NEB/T4 gene 32 page indicating many similar applications involving reverse transcriptases, polymerase etc.

"Recently, it has been shown to improve restriction enzyme digestion (6), improve the yield and efficiency of reverse transcription (RT) reactions during RT-PCR (7-9), enhance T4 DNA polymerase activity (10-11), as well as increase the yield of PCR products (12)."

EDIT: Although the reference (6) cited by NEB does not have any evidence that proves the enhancement of restriction digestion with single strand binding protein 32. This chapter might be helpful? - Kowalczykowski et al., "21 single-stranded DNA binding proteins." The enzymes. Vol. 14. Academic Press, 1981. 373-444.

From their abstract, it looks like the mechanism of enhancement could be through prevention of ssDNA hairpins so that the endunuclease can access it's recognition site (I haven't read the full chapter). Another fact supporting this is that ref(6) mentions that other interacting enzymes can work alongside SSBs only when SSB's are not saturating all the DNA in the in-vitro reaction. In an in-vivo context, the chapter abstract states that intracellular SSBs are sufficiently abundant to saturate all ssDNA intermediates formed inside cells (I presume they are talking about E. coli)

Below is the supporting text from reference (6) mentioned by NEB - bolding is mine, for emphasis : Bittner, M., R. L. Burke, and B. M. Alberts. "Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities." Journal of Biological Chemistry 254.19 (1979): 9565-9572.

Nuclease Assays-The presence of both exo- and endo- deoxyribonuclease contaminants in the 32 protein fractions has been monitored carefully throughout these purification schemes. The results of exonuclease assays are presented in Table III, and the results of sensitive endonuclease assays are presented in Table IV. Note that, when such assays are designed to detect nuclease activities on single-stranded DNA, it is important to test 32 protein at subsaturating, rather than only at saturating, protein to DNA ratios. Otherwise, an activity which hydrolyzes only free (non-32 protein-com- plexed) single-stranded DNA will escape detection.

  • $\begingroup$ Thanks for the response, that makes sense about dsDNA. I was mainly interested in the case of RE's cutting ssDNA, and how SSB's might interfere or not there. I looked at the paper in that is referenced in your quotation, but don't see the data that would point to the same conclusion. I assume it must be something in Table IV? $\endgroup$
    – etowah
    Mar 17, 2021 at 2:33
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    $\begingroup$ Oh yes. I couldn't find anything relevant in that paper, NEB might have cited it in error. I edited my answer with another potential reference $\endgroup$ Mar 18, 2021 at 0:51
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    $\begingroup$ I looked near both table III and IV $\endgroup$ Mar 18, 2021 at 1:03
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    $\begingroup$ Please do not post text as images. Copy and paste the text into your post. Images are not searchable, and can not be interpreted by screen readers for those with visual impairments. Use the edit link to modify your answer. See this for more information. Thanks! $\endgroup$
    – tyersome
    Mar 24, 2021 at 18:26
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    $\begingroup$ Ah, that makes sense. Thanks for pointing that out. I was lazy because it was an ancient PDF that wasn't copying well. I fixed it now :) $\endgroup$ Mar 26, 2021 at 6:04

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