Protein:DNA interactions

Question 1

Recognisable features of dsDNA for interaction?

Answer 1

Negatively charged phosphate backbone!

Base pair edges in major and minor grooves! (specific recognisable patterns for interaction with C-G and A-T base pairs)

Question 2

What can you recognise in the major groove that you can’t in a minor groove? of dsDNA

Answer 2

Which way around the base pair edges are, C-G vs G-C has symmetrical recognition features pattern in minor groove but asymmetrical in major groove.

Question 3

How do amino acids interact with individual bases?

Answer 3

Typically by hydrogen bonds, e.g.Arginine, R, to edge of Guanine in major groove. Or Asp/Glu (D/E) to exposed Guanine by pseudo-watson-crick

Question 4

Pi-stacking interactions?

Answer 4

Faces of base pairs interact, by aromatic pi stacking, excluding water. Aromatic amino acids and some other can interact with exposed base pair faces in this way.

Question 5

Role of lamba phage’s repressor protein, repressor c1?

Answer 5

Binds to bacterial DNA encoding phage (virus) DNA promoters, preventing virus expression in dormant stage of life cycle

Question 6

How does lamba phage repressor cI bind DNA?

Answer 6

Binds as a dimer at nearly palindromic site in cro promoter gene, each monomer on either side of palindrome axis of symmetry via major grooves. Exact size of dimer gives it shape complementarity with 2 major grooves. Most of interaction is sequence non-specific binding to sugar-phosphate backbone by part of HELIX TURN HELIX. Scans along backbone until HTH’s READING HELIX interacts in a sequence specific manner, H-bonding to base pair edges. (binding quite a long sequence 16bp despite being a small protein, this is advantageous because it makes the repressor very SPECIFIC for this cro promoter)

Question 7

What is helix turn helix motif?

Answer 7

HTH is a widespread DNA binding module. Contains sequence specific reading helix and sequence non-specific helix that interacts with DNA sugar phosphate backbone until reading-helix finds sequence.

Question 8

What is the function of the MetR, Met repressor protein?

Answer 8

Suppresses the production of methionine when S-adenosyl methionine levels are already high. By binding to dsDNA to block promoter sequence access. Uses BETA RIBBON to bind one major groove! (not HTH)

Question 9

How does Met repressor bind dsDNA?

Answer 9

As a dimer, unusually using a beta ribbon to bind a palindromic sequence in a single Major groove.

Question 10

What is the role of bacterial restriction enzymes (restriction endonucleases) in antiviral innate immunity?

Answer 10

Recognition of non-self, viral, DNA sequences inserted into the bacterial DNA, and excision of these sequences.

Question 11

How do Restriction enzyme containing E.coli strains protect their own DNA from being bound by EcoR-I (restriction endonuclease)?

Answer 11

They METHYLATE the R1 target sequences GAATTC. So only the newly inserted viral DNA, lacking these modifications (Methyl groups) will be recognised.

THis is done by a methyltransferase enzyme.

Question 12

What palindromic base sequence does EcoR1 recognise? And what does it leave when it cuts it?

Answer 12

GAA-TTC Sticky-ends are left, a 4base 5’ overhang 5’AATT……

Question 13

What is EcoR1 dependent on to be able to cut DNA at GAATTC sequence to leave its 5’AATT…. overhang?

Answer 13

Mg2+, magnesium required for enzyme action. Can bind but can’t cut.

Question 14

How does EcoR-1 recognise the GAATTC sequence?

Answer 14

By attempting to distort the DNA structure, inserting an a HYDROPHOBIC ALANINE residue between stacked A and T base pairs in the middle of the sequence. (A-T pairs will distort whilst the G-C’s interactions would be too strong/resistant to distortion) Simultaneously one ASPARAGINE (Asp, N) residue recognises adjacent T-T step in sequence by H-bonding to both T’s (also requiring distortion) WATER also mediates interactions between residues and DNA bases.

Question 15

Why is the product of EcoRV (a related restriction endonuclease to EcoR-1) not useful in lab? How does it interact?

Answer 15

Produces blunt ended fragments as its cuts its palindromic recognition sequence in the middle. Again it induces distortions in the DNA structure to test if it is the right sequence. Inducing distortion at the minor groove and binding sequence-specifically at the major groove to basepair edges. DNA so distorted if bendable AT step present that CONSECUTIVE bases can h-bond together! (again ‘distortability’ tested by restriction enzyme to find specific sequence) (typically pi-stacking between A and T weaker and more distortable than between G and C)

Question 16

How does M.Hha1, the methyltransferase in Haemophilus haemolyticus protect H.haemolyticus’ own GCGC palindromes from being cut by its restriction endonuclease?

Answer 16

It methylates the cytosine residues, which prevents the restriction endonuclease from recognising the site.

Acting as a monomer it methylates the cytosine on each strand one at a time, typically because it will encounter existing hemi-methylated sites.

Uses methyl groups from S-adenosylmethionine.

Question 17

Why does M.Hha1 methyltransferase encounter hemi-methylated GCGC sequences most often?

Answer 17

Because methylation is a lasting modification, and so one strand is from the parent DNA and so already methylated on C5 position, one is newly sythesised and so lacks methylation.

Question 18

What mechanism does M.Hha-1 methyltransferase use to methylate each cytosine in its target GCGC palindrome sequence?

Answer 18

Acting as a monomer it **flips out **one cytosine nucleotide, breaking watson-crick interaction. Leaving widowed Guanine residue. (rest of DNA largely undistorted)

Forms transient covalent bonded intermediate between Cysteine thiol group and cytosine.

Widowed Guanine also “recognised” by a loop of the enzyme that temporarily replaces cytosine.

Question 19

How can guanines be methylated, and why is this a problem?

Answer 19

Guanines are O6-methylated randomly by SAM (s-adenosylmethionine)

This is a problem because O6-methylated Guanine (^me6G) makes a better complementary match for Thymine during replication.

Therefore when replicating, the ^me6G ends up being replaced by an Adenosine.

Question 20

How are O6-methylated guanines, ^me6G, (alkylation damage by SAM) fixed?

Answer 20

By Alkyl guanine transferase, AGT.

A “suicide enzyme” with cysteine in its active site. Transfers methyl group from guanine to thiol group.

Can only do one reaction in its lifetime!

Flips out O6-methylated guanine. Transfers methylgroup to thiol group (SH) on cysteine residue.

Stabilises duplex DNA by interacting with widowed Cytosine by inserting Arginine into gap, which interacts.

Question 21

Why does the HTH (helix turn helix) motif in AGT (alkylguanine transferase) have a sequence **non-specific **reading helix?? (with hydrophobic, non-polar aas, like glycine alanine, G,A)

Answer 21

“Smooth greasy reading head” lol

Not sequence specific so that it can bind anywhere on DNA where O6-methylated guanines are.

Only has one arginine to “recognise” widowed Cytosine after flipping ^me6G out.

Question 22

How can alkyl-guanine transferase, AGT (suicide enzyme) recognise alkylations bigger than methyl groups on O6 of guanine?

Answer 22

Uncontrained, large, Modification pocket can fit a lot of shapes.

Question 23

What does UV light/irradiation do to DNA?

Answer 23

Two possible thymine photodimers formed.

Electrons excite the 5,6 C=C double bond in consecutive thymines (also works with other pyrimidine pairs)

Causes formation of cyclobutane photodimer!

(consequentially the deformation this causes prevents complementary pairing with the second thymine during replication, causing a deletion of an adenine, a frameshift mutation)

A 6-4 photodimer between adjacent thymines is another possible consequence, a transfer of an activated oxygen between thymines, causing them to covalently bond together, grossly distorting the structure and preventing DNA polymerase activity entirely.

Question 24

What repairs thymine photodimers (particularly 6-4) in drosophila melanogaster (fly)?

(and what repairs general damage in humans?)

Answer 24

Drosophila has a 6-4 **photolyase **enzyme that uses UV light energy (harvested via deazaflavin, electron provided by FADH) to reverse the original damaging reaction.

It finds thymine photodimers by attempting to insert a protein wedge into DNA duplex to displace poorly paired and poorly stacked bases.

(XP complex repairs lots of stuff in humans)

Question 25

How does Drosophila melanogaster (6-4) Photolyase find and repair thymine (pyrimidine) dimers?

(how does it collect light?)

Answer 25

Inserts protein wedge into dsDNA helix, pushing out only the poorly stacked and paired thymine dimers.

The wedge non-specifically compensates/stabilises DNA structure for flipping out thymine dimers by binding to phosphate backbone. Widowed bases themselves are left unpaired.

Flipped out thymine dimer enters binding pocket in lyase where it receives free electron from FADH cofactor. Eventually the bonds arrange themselves correctly again, and the photolyase releases.

Uses deazaflavin chromophore to harvest UV light energy. passes photons to FADH which releases an electron.