Lecture 6: Restriction and modification enzymes

Question 1

T/F: different types of enzymes can modify DNA

Answer 1

true!!

Question 2

________: enzymes that break phosphodiester bonds i.e. DNA!

Answer 2

phosphodiesterases

Question 3

______ - remove bases starting at ”ends” of linear DNA or
gaps/nicks in circular DNA
examples: (in gel, linear vs circular)

Answer 3

Exonucleases

Question 4

why do bacteria have a TON of exonucleases?

Answer 4

mainly for defense against phages- if we chew up linear phage DNA we can protect ourselves!

Question 5

_______ can be specific or non-specific

Answer 5

Endonucleases

Question 6

_____ endonucleases: restriction enzymes cut dsDNA at specific
target sites [restriction sites]

Answer 6

specific

Question 7

_____ endonucleases – referred to as DNases (DNAses)
(deoxyribonucleases) or RNases RNAses

Answer 7

nonspecific

Question 8

what are the specific endonuclease systems?

Answer 8

Restriction (R,
endonuclease) and Modification (M, methylase)
systems in bacteria = R/M

Question 9

______ applies to sequence specific nucleases that
break nucleic acids chain within the molecule rather than
at the ends

Answer 9

Endonuclease

Question 10

R/M systems act as defense against _____ and are widespread in Bacteria

Answer 10

foreign DNA

Question 11

explain the R/M system:

Answer 11

protect resident DNA against contamination by
sequences of foreign origins i.e. phages

”strategy” it to modify your own DNA so it doesn’t get
cut, and “restrict” non-modified (i.e. foreign) DNA

“restriction” – phage replication is “restricted” to hosts
that don’t have an R/M system

Question 12

T/F: Restriction enzymes can’t bind
restriction sites that are
modified by methylation

Answer 12

true

Question 13

why is it significant that phage DNA cannot be methylated?

Answer 13

restriction enzymes can only cut sites without methylation- we methylat our DNA so that we can protect our own DNA

Question 14

____ (modifies A) in GATC
and _____ (modifies C) CmCWGG
systems in E. coli are the most
important

Answer 14

dam
dcm

Question 15

One of the best-studied DNA base modifications is _______

Answer 15

5-methylcytosine (5mC)

Question 16

explain 5-methylcytosine (5mC) as a base modification

Answer 16

DNA methyltransferases (Dnmts)→ transfer a methyl
group from S-adenosylmethionine (SAM) to the fifth
position of cytosine

Question 17

________:
-base to be methylated
is “flipped out” so that
methyl-group can be
added to a C at GCGC

Answer 17

HhaI methylase

Question 18

_______ – most use SAM = S-adenosyl-methionine as a CH3-donor

Answer 18

Methylases

Question 19

_______:
occurs when a nucleic acid-processing enzyme needs access
to the base to perform work on it, such as its excision for
replacement with another base during DNA repair, in addition to
methylation, and replication
* It can also occur in RNA double helices or in the DNA:RNA
intermediates formed during RNA transcription

Answer 19

base-flipping

Question 20

what is the most common type of restriction enzyme?

Answer 20

Type IIP, cleaves symmetruc targets and cleavage sites

Question 21

MOST ENZYMES USED IN LAB ARE…

Answer 21

Type II REs

Question 22

Type II restriction enzymes cut at…

Answer 22

predictable, specific target sites (usually PALINDROMES!)

Question 23

some REs generate blunt ends (like ____) and others make sticky ends (like _____)

Answer 23

SmaI
EcoRI or KpnI

Question 24

how do we classify RE?

Answer 24

• Enzyme complexity
  – Cofactor requirements
  – Position of DNA cleavage

Question 25

For all types _____ is the methyl donor even though the structure of the methylase may differ

Answer 25

SAM- S adenosyl-methionine

Question 26

T/F: all four types of restriction enzymes will not cut hemi-methylated DNA (one strand only)

Answer 26

true!

Question 27

hemi-methylated target sites are substrate for methylation by _______

Answer 27

Dam/Dcm methylases

Question 28

non-methylated target sites induce _______ activity

Answer 28

endonuclease

Question 29

_______– recognition sequence is far from cleavage site (1000 bp) - modification of recognition site by methylation prevents cleavage

Answer 29

TYPE I RE

Question 30

what is the subunit formation of type I REs?

Answer 30

the R, M, and S subunits are all connected together

Question 31

A model of DNA translocation for type DNA I restriction enzymes is...

Answer 31

Looping (requires active Translocation) and requires ATP winds DNA through itself and pulls on it- then once DNA is pulled far enough- it cuts

Question 32

________ are members of a large complex class that has been further divided into six subclasses based on the specificity of sequence recognition, the cleavage reaction, and methylation sensitivity

Answer 32

Class II restriction endonucleases

Question 33

Type IIP = ________

Answer 33

palindrome type

Question 34

Type IIS = _______

Answer 34

shifted cleavage

Question 35

When Type IIS enzymes bind to DNA, the catalytic domain is positioned to one side of, and several bases away from, the sequence bound by the recognition domain, and so cleavage is ‘shifted’ to one side of the sequence. IIS enzymes may not require _________

Answer 35

palindromic sequences

Question 36

what is the recognition of Type II RE?

Answer 36

cleavage site and site of methylation, cutting if there's no methyl group

Question 37

_______ palindrome type: Cut right at palindromic binding site to give blunt or sticky ends, depending on the enzyme

Answer 37

Type II RE

Question 38

what is the general structure of Type II RE?

Answer 38

R+S subunit and an M+S subunit connected together

Question 39

what's the difference between Type IIS and Type IIG subunits?

Answer 39

Type IIS: R+S, M+S Type IIG: R+M+S

Question 40

what is the recog. site of Type III RE?

Answer 40

recognition sequence is made of 2 “inverted repeats” in same DNA -cleavage is away from the site

Question 41

what's the subunit of Type III RE?

Answer 41

R+M+S

Question 42

_______ restriction enzymes (act in a similar way to type II G) BUT cut modified target sites that are methylated or glucosylated (part of “war” with phage – some phage try to “hide” their cut sites with sugar molecules – some bacteria develop enzymes that recognize these modified sites)

Answer 42

Type IV

Question 43

why do Type IV cut at sites that are methylated? wouldn't that be dangerous to us?

Answer 43

some phage try to “hide” their cut sites with sugar molecules – some bacteria develop enzymes that recognize these modified sites... i.e. Type IV!!

Question 44

what is the subunit formation for Type IV RE?

Answer 44

either R+M and S or R+M+S

Question 45

what are the applications for RE? mostly for type II

Answer 45

Restriction fragment length polymorphism (RFLP) – i.e. sampling for sequence diversity Physical mapping and confirming DNA Generating DNA fragments for cloning

Question 46

why would we use RFLP for screening lots of alleles... over sequencing?

Answer 46

because its faster and cheaper- especially when we're examining lots of alleles

Question 47

what is the procedure for using RE for physical mapping of DNA?

Answer 47

Physical maps for DNA - cut DNA into smaller fragments with restriction enzymes and resolve by agarose gel electrophoresis - visualize DNA on agarose gels by 1. Staining with ethidium bromide OR 2. Southern blot analysis if “low copy” number (relatively archaic now that sequencing/PCR exists)