Nucleases and DNA Ligase

Two ways to classify nucleases

A nuclease hydrolyzes phosphodiester bonds ¹. Name one by both where it cuts and what it cuts:

Exonucleases have a direction (5’→3’ or 3’→5’) and need an accessible end; endonucleases don’t.

Restriction endonucleases

Bacteria make restriction enzymes to chop up invading phage DNA, protecting their own DNA by methylating the same sites. Their lab value is specificity: each cuts only at its own 4- to 8-base recognition site ³. Sites are usually palindromes - both strands read the same 5’→3’.

• Sticky (cohesive) ends come from staggered cuts. Any two ends made by the same enzyme have complementary overhangs and can pair ³.
• Blunt ends come from cuts straight across; any blunt end joins any other, but less efficiently.

A 6-base site occurs on average once every 4⁶ = 4,096 bases in random sequence; a 4-base site once every 256.

The order and spacing of sites is a restriction map. A variant that creates or destroys a site changes fragment sizes - RFLP analysis. The classic case: the sickle cell change in HBB (GAG→GTG) destroys a site for the enzyme MstII (CCTNAGG), so the normal gene cuts into two fragments and the sickle allele stays as one longer fragment. Today the same idea is used after PCR (PCR-RFLP): amplify a short region, digest, and read the bands on a gel.

Cleanup nucleases

Nonspecific nucleases are cleanup tools during nucleic acid prep:

• RNase A digests co-extracted RNA out of a DNA prep. In RNA work it’s the enemy: RNases are on skin and dust, survive autoclaving, and refold after boiling, so RNA work uses RNase-free tubes and tips, gloves, and RNase inhibitors ³.
• DNase I removes genomic DNA from an RNA prep before RT-PCR, so DNA isn’t amplified and counted as RNA. It must be removed or heat-inactivated before reverse transcription.
• RNase H cuts the RNA strand of an RNA:DNA hybrid. Many reverse transcriptases carry it; after cDNA synthesis it clears the RNA template. It also drives some isothermal amplification methods.
• Uracil-N-glycosylase (UNG) isn’t strictly a nuclease, but it removes uracil from DNA, leading to strand breaks. If a lab makes all its amplicons with dUTP instead of dTTP, adding UNG to each new reaction destroys any leftover amplicon before cycling starts; real templates contain T and are untouched, and the 95 °C step kills UNG. It’s a standard defense against PCR carryover contamination.

DNA ligase

DNA ligase remakes a phosphodiester bond between a 3’-OH and an adjacent 5’-phosphate, sealing a nick in double-stranded DNA ¹. In cells it joins Okazaki fragments and finishes repairs ². T4 DNA ligase (from a phage, powered by ATP) is the standard lab enzyme; thermostable ligases are used in ligation assays that cycle temperature.

Ligase needs two things ³:

1. the right chemistry - a 3’-OH facing a 5’-phosphate; remove the phosphate (with a phosphatase) and the end won’t ligate, a trick that stops a cut vector from closing on itself;
2. the ends held together - by paired sticky overhangs, or by blunt ends meeting.

Cloning. Cut vector and insert with the same enzyme, mix, and ligase seals the matching ends into a recombinant plasmid ³.

Ligation-based genotyping. Two probes bind side by side on the target, meeting exactly at the variant position. Ligase joins them only if the junction is perfectly paired; a mismatch leaves a gap ligase won’t seal. The oligonucleotide ligation assay (OLA) turns “is the sequence there?” into “is there a joined product?” ³. MLPA, which counts exon copy numbers, is built on the same principle.

What to take from this

Nucleases are exo (from an end) or endo (internal), and DNase or RNase. Restriction enzymes cut palindromic sites into sticky or blunt ends, and gained or lost sites change fragment sizes (RFLP; sickle cell destroys an MstII site). RNase A cleans DNA preps, DNase I cleans RNA preps, RNase H removes RNA from hybrids, and UNG destroys dUTP-containing amplicons to prevent carryover. Ligase seals a 3’-OH to a 5’-phosphate when the ends are held together, enabling cloning and mismatch-sensitive ligation assays like OLA and MLPA.