[2S] UNIT 5 Characterization of Nucleic Acid

Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.

NUCLEASES SPECIFICITY Specific for DNA

NUCLEASES SPECIFICITY • Targets RNA • Degrades all RNA

NUCLEASES SPECIFICITY • Able to cleave DNA and RNA hybrid • Subtype of RNase

NUCLEASES SPECIFICITY Degrades RNA bound covalently to DNA

When a nuclease hydrolyzes an ester bond in a phosphodiester linkage, it will have a specificity for either of the two ester bonds. This generates what nucleotides?

For single strand molecules (RNA), ______ can rapidly degrade RNA molecules into ribonucleotide subunits.

TWO TYPES OF NUCLEASE Hydrolyze internal bonds within a polynucleotide chain

TWO TYPES OF NUCLEASE ● Cut the length of the DNA sequence ● Break internal phosphodiester bonds

TWO TYPES OF NUCLEASE Cut at any point depending on its target site ○ Usually in the middle portion of the fragment ○ Between the 5’ and 3’ terminus

TWO TYPES OF NUCLEASE Produce several segments of our polynucleotide

TWO TYPES OF NUCLEASE Target and remove the terminal nucleotide

TWO TYPES OF NUCLEASE Removes nucleotides one at a time from the end of a DNA molecule

TWO TYPES OF NUCLEASE Cuts the terminal nucleotide whether it is on the 5’ or 3’ (only at the end of the fragment)

MECHANISM OF NUCLEASE HYDROLYSIS Removes nucleotides from both strands of a double-stranded molecule

MECHANISM OF NUCLEASE HYDROLYSIS Progressive shortening of the dsDNA from both ends after treatment

MECHANISM OF NUCLEASE HYDROLYSIS ● Removes nucleotides from the 3’ terminus ● Can only cut through double strands

MECHANISM OF NUCLEASE HYDROLYSIS Cleaves only single-stranded DNA, including single-stranded nicks in mainly double-stranded molecules

MECHANISM OF NUCLEASE HYDROLYSIS Can only cut single strands nucleotides ○ Create a nick in our double stranded nucleotide ○ Cannot entirely cut the sequence (double stranded)

MECHANISM OF NUCLEASE HYDROLYSIS Refers to a specific type of discontinuity

MECHANISM OF NUCLEASE HYDROLYSIS You need another digestion for that exposed single strand to fully cut the segment ○ However, if your DNA is single stranded, it can entirely create fragments

MECHANISM OF NUCLEASE HYDROLYSIS Cleaves both single and double-stranded DNA

MECHANISM OF NUCLEASE HYDROLYSIS Depending on which part it attaches to or targets ○ Can cleave segments or several segments

MECHANISM OF NUCLEASE HYDROLYSIS Produce mononucleotide