30 - B cells: Generation of BCR diversity

Question 1

Primary diversity (primary diversification)

Answer 1

Through process of somatic recombination – get primary diversity, including combinatorial diversity & junctional diversity events

Question 2

Heavy chain & light chain gene families are encoded on

Answer 2

separate chromosomes

Question 3

Variable region of Ig - Ig gene diversity: somatic recombination

Answer 3

B cells use groups of segments of genes = create different possible Ab using somatic recombination

Somatic recombination: process segments are rearranged

Tightly regulated machinery controls recombination processes

Many of the proteins are involved in DNA repair function

Question 4

Light chain 3 region genes

Answer 4

o Variable region gene (V)
o Joining region gene (J)
o Constant region gene (C)

Question 5

Heavy chain 4 region genes

Answer 5

o Variable region gene segments (V)
o Diversity region gene segments (D)
o Joining region gene segments (J)
o Constant region gene segments (C)

Question 6

CDR 1 & 2 = encoded in

Answer 6

V segments of light & heavy chains

Question 7

CDR3 encoded in

Answer 7

most variable CDR
-encoded in:
Joining of V-J segments of light chain
V, D, J gene segments of heavy chain

Question 8

Variable region of both heavy & light chain = composed of…

Answer 8

…segments from recombined V, D (only H chain) & J genes

Many different V, D, J segments in germline DNA

-we Inherit all segments/options

Question 9

when does somatic reocmbination occur?

Answer 9

During B cell development
-V(D)J recombination occurs to choose one of each to make up the variable region

Question 10

how does light chain adds to diversity?

Answer 10

2 different loci – on separate chromosomes with different constant regions
-Only 1 will be expressed per BCR in light chain of Ab
 κ chain
 λ chain

-Which one gets activated first inhibits the other one
-Only one chain will be expressed & will silence the other

each locus includes many different V & J regions

• 38 V regions to choose from
• 5 J region to choose from

Question 11

how does heavy chain adds to diversity?

Answer 11

1 locus

Include many different V, D, J regions

Many different constant regions represent different isotypes
-IgM, IgG, IgD, etc…

• 46 V regions to choose from
• 23 D regions to choose from
• 6 J region to choose from

Question 12

mechanism of recombination event

Answer 12

1. Inherited – stem cell
   -Precursor B cell
2. Somatic recombination – take one of each segment
3. Recombinase proteins join separate gene segments together
4. transcription, need mRNA and translated into protein

• make BCR of naïve B cell
  -Irreversible
  -Somewhat everything in between & not chosen = deleted

Question 13

Combinatorial diversity, what happens?

Answer 13

somatic reocmbination

Diversity – resulted from different combinations of V,D, J regions

Question 14

Combinatorial diversity, RSS

Answer 14

RSS (Recombination signal sequences)
-flank each Ab gene segment
-in front or downstream of all of these V, J segments,

Recombinase enzymes recognize the RSS
-Each has a conserved nonamer (9bp) & heptamer (7bp) sequence
-Either a 12- OR a 23-bp spacer sequence lies between nonamer & heptamer

Spacing & arrangement dictate that a 12-bp RSS must pair with a 23-bp RSS for recombination to occur
-12/23 Rule

Question 15

RSS -12/23 Rule

Answer 15

Spacing & arrangement dictate that a 12-bp RSS must pair with a 23-bp RSS for recombination to occur

Question 16

Recombination signal sequences (RSS) direct …

Answer 16

…pairing of different segments together
* Light chain: V-J
* Heavy chain: D-J and then V-DJ
* RSS of VH has 23 bp spacer
Segments in between selected ones will be deleted

Question 17

Recombination signal sequences (RSS) allow…

step by step (4)

Answer 17

…looping of DNA & binding by specific proteins

1. RSS regions are brought together creating loop in DNA
   -Loop part contain segments that were not selected
2. RAG= recombination activating gene
   -RAG-1 & RAG-2 = necessary for recombination (Responsible for recognizing & cutting DNA at immunoglobulin-encoding region & the RSS)
   -Covalently closed DNA hairpin ends
3. Loops is excised (Signal joint)
   -No longer on chromosomes = deleted
4. Coding region of selected V & J regions remain (Coding joint)

Question 18

Signal joint

Answer 18

Loop that contains all of those segments in the middle that were not selected is called the signal joint.

It gets excised and gets deleted.

Question 19

Coding joint

Answer 19

Contains the selected segments.
ones that were combined together, then going to code for the light chain for that BCR

Question 20

Kappa & lambda refers to

Answer 20

DNA segment code for light chain

Question 21

Junctional diversity – additional variety

Answer 21

During recombination, nucleotides may be added/removed at junctions between V&D, D&J or V&J for light chain

Question 22

what happens at Junctional diversity in general

Answer 22

-Signal joint is ligated together & discarded

-At coding ends: repair proteins bind the hairpin

-Artemis: endonuclease = Opens (nicks) the covalently closed DNA hairpins

Question 23

Junctional diversity: Hairpin cleavage:

Answer 23

Happens mainly in Light chain

Hairpin can be opened in3 different ways by Artemis

-Additional P (palindromic) nucleotides at overhangs

-OVERHAND = Template –> allowing DNA repair enzymes to fill in complementary strand

-Complementary strands of DNA read the same in both directions (5’end or 3’end)

Question 24

Hairpin can be opened in3 different ways by Artemis

Answer 24

-having an overhang at the bottom
3’–>5’

-nick it in the middle where it’s a little blunt
- an overhang up here from the
5’–>3’

depending on where it Nicks = most likely going to be an overlap overhang.

Question 25

Junctional diversity: Exonuclease activity

Answer 25

mostly in heavy chain may remove nucleotides on each side of the coding joint TdT (Terminal deoxynucleotidyl transferase) can add up to 20 N-nucleotides (non-template-encoded) to cleaved strands Repair enzymes then trim off nonmatching nucleotides -Fill in remaining single-stranded gaps & ligate the new DNA

Question 26

Reason for why CDRs vary in length

Answer 26

TdT can add up to 20 N-nucleotides

Question 27

Junctional diversity: summary

Answer 27

During recombination -- nucleotides may be added/removed at junctions between V&D, D&J (or V&J for light chain) if there arent much template there: -TdT (Terminal deoxynucleotidyl transferase) -Adds nucleotides to cleaved strands -Primarily in heavy chains -Unpaired nucleotides=removed by exonuclease

Question 28

Mechanism of V(D)J recombination

Answer 28

Mechanisms generate BCR diversity in naive B cells Multiple gene segments -gene segments are put together -Heavy/light chain combinatorial diversity Junctional diversity: P nucleotide addition -Templated nucleotide addition between joints =resulting from asymmetrical cleaving of hairpin structures Exonuclease trimming -Sometimes occurs at junctions – lose nucleotides Nontemplated N nucleotide addition -Mediated by TdT activity -Adding in random nucleotides between joints --due to exonucleases that can trim a bit too much

Question 29

Combinatorial diversity

Answer 29

 Mechanism involves RSS sequences being brought tgt  RAG-1/2 crucial proteins signal joint & coding joint  Loops excised (signal joint) & removed while coding joint remains

Question 30

Junctional diversity

Answer 30

 TdT protein inserts nucleotides at junctions - create even more diversity  Exonucleases removing nucleotides

Question 31

Primary diversification

Answer 31

o Combinations of H & L chains o Junctional diversity o Combinatorial diversity

Question 32

Deficiency in .., an enzyme involved in V(D)J recombination would result in decreased N-nucleotide addition to V-DJ & D-J heavy chain junctions

Answer 32

if no TdT = unable to add non-template nucleotide at VJ & DJ junction segments

Question 33

BCR diversity proteins involved

Answer 33

RAG1/2 Artemis Exonuclease -Remove nucleotides not matching complementary strand TdT -Add non-template nucleotide

Question 34

BCR diversity step-by-step

Answer 34

1. RSS that are flanking each of these segments. 2. RAG1/2 recognize those RSS & brought together -- then cleaved off. -signal joint that is deleted, removed 3. Nicks that are made by Artemis. - Artemis can nick the covalent DNA hairpin and depending on where it's nicked, either there's some overhang. 4. if there's some overhang= template where just DNA repair mechanisms can add the complement nucleotide to it, leading to P nucleotides. 5. possibility of extensive trimming at the D junction, in the H chain. 6. don't really template, so need to add non template nucleotides( random nucleotides) until there's some sort of junction taking place.