BIOL 2P02 Lecture Notes - Lecture 3: Sticky And Blunt Ends, Restriction Enzyme, Puc19
21 May 2018
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Spring BIOL 2P02 Lecture 3 17/05/2018
Chapter 6 Continued
• DNA is double stranded
o Nucleotide is wedge shaped in 3D space
▪ As they join it forms a circle -> creates a spiral helix
▪ Naturally twists
• E- cloud takes up space
• Major Groove & Minor Groove of DNA
o
▪ > 180 degrees for the major groove
▪ <180 degrees for the minor groove
o Hypothetical Situation
▪ The 2 grooves would have similar size if dR residues were attached at 180º to
each other
▪
• No major and minor grooves -> the grooves would be the same size
o
o H bond donors & acceptors in DNA grooves facilitate its recognition by proteins
▪ The edges of base pairs displayed to DNA major & minor groove contain
potential H-bond donors & acceptors
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find more resources at oneclass.com
Spring BIOL 2P02 Lecture 3 17/05/2018
▪ For a protein to bind to a DNA strand it needs to bond -> needs a H bond
▪ Two types of DNA binding proteins
• Specific & non-specific
o Specific has a pocket that attracts DNA -> binds to a very
specific sequence i.e. ATTC
o Non specific will bind anywhere
• How do they bind
o Each side can form H bonds with the amino acid sof a protein
o Some may be H bond acceptor
o Some may be H bond donor
▪
o Enzymes can read or sequence the DNA without modification of
the strand by binding to the major or minor groves
▪ Will read and move along the strand until they find the
sequence they need
▪ Ezyes look fo speifi pattes of:
▪ 1)H bond acceptors (A)
▪ 2)H bond donors (D)
▪ 3)nonpolar (H)
▪ 4)methyl groups(M) -> thymine
▪ Nonpolar groups on cytosine
▪ major groove can provide exact base pairs
▪ minor grove can only determine whether there is an
AT/TA or GC/CG base pair
▪ comparing majhor to minor
• CG and GC are different -> major groove
• minor groove are the same
o it cannot tell the difference between which is on which side
o
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Spring BIOL 2P02 Lecture 3 17/05/2018
▪ The major groove and not the minor can nid to DNA binding proteins.
True/False
• False
• Restriction endonucleases
o Specific DNA binding proteins
▪ Come from bacteria/archaea
▪ Recognize a specific DNA sequence and snip it
▪ Some leave sticky ends (overhang)
• Allows for joining of other things
o Blunt restriction enzyme
▪ No sticky ends
o
▪ Derived from an E. coli strain and recognizes the GAATTC and cuts at it
▪ How is it recognized
• The protein has a domain that will recognize the DNA
▪ Why do ateia/ahaea hae these ut e do’t?
• For viruses
o They inject stuff into the bacteria/archaea
o This is a defense system so bacteria can recognize the virus
o What pattern is seen?
▪
• Palindromic
o Same going in each direction
▪ Allows for the same sticky end on both sides
▪ otato akads is otato
▪ ’ GAATTC 3’
▪ 3’ CTTAAG 3’
• Palindromic
o Most common recognize short palindromes
o Cleavage occurs at asymmetrical positions w/n this sequence produces fragments w/
oehagig ’ o 3’ eds
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find more resources at oneclass.com
Document Summary
At/ta or gc/cg base pair comparing majhor to minor: cg and gc are different -> major groove, minor groove are the same it cannot tell the difference between which is on which side. 17/05/2018: the major groove and not the minor can nid to dna binding proteins. Can you think of a problem with this approach and/or advantage(s) to using two restriction enzymes instead: sticky ends match- they could attach. 17/05/2018: how to fix it, ce(cid:396)tai(cid:374) te(cid:373)pe(cid:396)atu(cid:396)es so they do(cid:374)"t (cid:396)ea(cid:374)(cid:374)eal, use to different restriction enzymes, prevents upside down, cut with two different restriction enzynes, gives asymmetric , unmatching sticky ends, the restiruction enzyme bamhi cleaves the sequence. No: can form many structures within it. Inverted repeats: a sequence in the dna that is following downstream by its reverse compliment, not a palindrome. If single stranded it can fold into a hairpin (tiny) or stem (large: stem is the inverted repeate, 6 base pairs.
