DNA Recombination and DNA Repair

1. What is recombination?

Breaking and joining of DNA molecules

2. What are two different types of recombination?

1) Homologous recombination

2) Nonhomologous recombination

3. What is the important requirement for homologous recombination?

Nucleotide sequences must be exchanged between two similar or identical molecules of DNA (chromatids) during meiosis.

4) During which stage of the cell cycle does homologous recombination occur?

Prophase I

5) What is the difference between sister and nonsister chromatids?

Sister Chromatids

• Definition: Two identical copies of a single chromosome, created during DNA replication.

• Origin: Come from the same chromosome.

• Relationship: Genetically identical (except for rare replication errors).

• Example: The two halves of a duplicated chromosome (X-shape).

---

Nonsister Chromatids

• Definition: Chromatids from homologous chromosomes (one from mom, one from dad).

• Origin: From different chromosomes in a homologous pair.

• Relationship: Similar but not identical—have the same genes but possibly different alleles.

• Role in meiosis: Crossing over happens between nonsister chromatids during prophase I of meiosis.

What is the term?

Defintion: Association between two homologous chromosomes

synapsis

What is the term?

Defintion: Two replicated chromosomes paired up

Tetrad/Bivalent

What is the term?

Defintion: A higher order structure that forms after synapsis

synaptonemal complex

What is the term?

Definition: Point where crossover has occurred?

Chiasma

What is the term?

Defintion: Chromosomes that have a pair of sister chromatids

Replicated chromosomes

6) What is the difference between homologous chromosomes and nonhomologous chromosomes?

Homologous Chromosomes

• Definition: A pair of chromosomes—one from each parent—that carry the same genes in the same order, but may have different alleles.

• Example: Chromosome 1 from your mom and chromosome 1 from your dad.

• Structure: Same length, same centromere position, same gene loci.

• Function: Pair up during meiosis I for synapsis and crossing over.

• Chromosome number: Humans have 23 pairs of homologous chromosomes (46 total).

---

Nonhomologous Chromosomes

• Definition: Chromosomes that are not a pair—they carry completely different sets of genes.

• Example: Chromosome 1 and Chromosome 5.

• Structure: Different lengths, gene content, and centromere positions.

• Do they pair in meiosis? ❌ No — they do not undergo synapsis or recombination with each other.

• Function: Involved in different biological roles because they carry unrelated genes.

7) What is the difference between synapsis and crossing over?

🧬 Summary Table:

Feature	Synapsis	Crossing Over
What happens?	Homologous chromosomes pair up	Nonsister chromatids exchange genetic material
When?	Zygotene (Prophase I)	Pachytene (Prophase I)
Involves?	Whole chromosomes	Sections of chromatids
Leads to?	Formation of tetrads	Formation of recombinant chromatids
Increases variation?	❌ No (just alignment)	✅ Yes

8) Recombination is the breakage and reunion between which two structures?

Nonsister chromatids (usually) of homologous chromosomes

9) What are the chiasmata?

Point where crossover has occurred

10) In prokaryotic systems, is an enzyme needed to cause a double-stranded break? What about in eukaryotes?

🦠 In Prokaryotes (like bacteria):

• Double-stranded breaks are not usually required to initiate homologous recombination.

• Instead, recombination often begins with a single-stranded gap or nick.

• Enzyme needed?
  ❌ Not necessarily for DSB initiation — it often starts from nicks, gaps, or existing DNA damage.

---

🧬 In Eukaryotes:

• YES — a double-stranded break is required to initiate homologous recombination, especially during meiosis.

• This DSB is deliberately introduced by a specific enzyme.

• Key enzyme:

  • Spo11 — creates intentional DSBs in meiotic prophase I.

• These breaks are then processed to allow strand invasion and crossing over between nonsister chromatids.

• Enzyme needed?
  ✅ Yes — Spo11 (and others) are required to initiate the recombination process.

11) In what ways is spo11 similar to Topoisomerase II?

🧬 Summary:

• Both Spo11 and Topoisomerase II use a tyrosine-based mechanism to create double-stranded breaks and form covalent intermediates with DNA.

• Spo11, however, does not rejoin the DNA it cuts — its purpose is to start recombination, not resolve topological stress.

12) Why would a virus integrate itself into a host instead of multiplying itself?

🧬 Summary:

Reason	Benefit
Latency	Avoids immune detection
Long-term persistence	Waits for favorable conditions
Access to host machinery	Especially important for retroviruses
Host preservation	Prevents early host cell death
Vertical transmission	Inherited by offspring in some cases

13) What is the minimum length of a homology needed for site-specific recombination to occur?

14-50 nucleotides

14) Given that Cre is an integrase and LOX are short target sequences, what type of recombination would you expect to take place?

🧪 Summary:

• Type of recombination: ✅ Site-specific recombination

• Enzyme: Cre recombinase

• Target sequences: LoxP sites

• Used in: Genetic engineering, transgenic mouse models (e.g., conditional knockouts)

15) Which of the following (attP, attB, attL, attR, IHF, Int, and Xis) are required for integration of a lambda phage?

✅ Required for Integration (NOT excision):

Component	Function
attP	Phage attachment site
attB	Bacterial attachment site
Int (Integrase)	Enzyme that catalyzes integration
IHF (Integration Host Factor)	Host protein that bends DNA to help the integrase function

These are all required for integration of lambda DNA into the E. coli chromosome.

16) Which of the following (attP, attB, attL, attR, IHF, Int, and Xis) are required for excision of a lambda phage?

✅ Required for Excision:

Component	Function
attL	Left hybrid site (formed during integration)
attR	Right hybrid site (formed during integration)
Int (Integrase)	Enzyme that catalyzes both integration and excision
Xis (Excisionase)	Accessory protein required only for excision; helps Int reverse the integration
IHF (Integration Host Factor)	Host protein that assists DNA bending for recombination

These are all required to recombine attL and attR, restoring attP and attB and excising the phage DNA.

17) Can phage lambda integrate into random locations in the bacterial chromosome?

No, phage lambda cannot integrate into random locations in the bacterial chromosome.

---

✅ It integrates at a specific site only:

• attB = the specific bacterial attachment site.

• Located in the E. coli chromosome, usually between the gal and bio operons.

• The phage lambda genome contains a matching site: attP.

---

🧬 Integration mechanism:

• Site-specific recombination between attP (phage) and attB (bacterium).

• Mediated by Int (integrase) and IHF.

• This specificity ensures precise and stable integration.

---

❌ Random integration?

• Not typical for lambda phage.

• Random integration might occur with some other viruses or under lab-induced conditions (e.g., when using engineered systems), but not in natural lambda phage infection.

18) What is an enzyme that is required for excision but not for integration of a lambda phage?

The enzyme that is required for excision but not for integration of a lambda phage is:

✅ Xis (Excisionase)

---

🧬 Role of Xis:

• Xis is a phage-encoded protein.

• It works alongside Int (integrase) and IHF (host factor) to mediate excision of lambda DNA from the bacterial genome.

• Specifically, Xis helps recognize and align the attL and attR sites for recombination.

• It also alters the activity of Int, shifting it from promoting integration to promoting excision.

---

🔁 Summary:

Enzyme	Required for Integration?	Required for Excision?	Notes
Int (Integrase)	✅ Yes	✅ Yes	Catalyzes recombination
Xis (Excisionase)	❌ No	✅ Yes	Needed only for excision
IHF	✅ Yes	✅ Yes	Host DNA-bending protein

19) Topoisomerase I is an enzyme that cuts one of the two strands of dsDNA, relaxes the strand, and reanneal the strand. How is this different than an integrase?

🧬 Topoisomerase I

Feature	Description
Function	Relieves supercoiling (torsional strain) in DNA during processes like replication and transcription

🔄 Integrase

Feature	Description
Function	Mediates recombination between two different DNA molecules (e.g., virus and host genome)

🧪 Summary Table:

Feature	Topoisomerase I	Integrase
Cuts DNA?	✅ One strand	✅ Both strands
Rejoins DNA?	✅ Yes, same strand	✅ Yes, joins different DNA molecules
Sequence-specific?	❌ No	✅ Yes
Role	Relieves supercoiling	Integrates DNA into genome
Example	Human Topo I	Lambda Int, HIV Integrase

20) What is the difference between the integrases Int from phage lambda and Cre from phage P1?

🧬 Key Differences Between Int and Cre:

Feature	Int (lambda integrase)	Cre (P1 recombinase)
Phage	Lambda (λ)	P1
Recombination type	Unidirectional, regulated integration/excision	Reversible, simple site-specific recombination
Cofactor requirement	✅ Needs host and phage proteins (e.g., IHF, Xis)	❌ No cofactors needed — works with just Cre and LoxP
Recombination sites	attP (phage) + attB (bacterium) → attL + attR	Two identical LoxP sites (34 bp)
Directionality control	Controlled by switching presence/absence of Xis	Not regulated — recombination depends only on LoxP orientation
Mechanism	Integrates or excises phage genome into/out of E. coli chromosome	Recombines any DNA between two LoxP sites (used in genetic engineering)
Used in research?	Occasionally	✅ Very widely used (Cre-Lox system for conditional knockouts)

---

🔬 In Summary:

• Int is more complex and requires host factors (e.g., IHF) and a helper protein (Xis) for excision.

• Cre is a standalone, simple enzyme that acts only at LoxP sites and is widely used in genetic engineering because it doesn't need any accessory proteins.

21) During the integrase mechanism, on the integrase enzyme, which end of DNA is attached to which amino acid?

🧬 Answer:

During integrase-mediated recombination:

The 5′ end of the cleaved DNA is covalently attached to a tyrosine residue on the integrase enzyme.

---

🔬 Explanation:

• Integrases (like lambda Int or Cre recombinase) use an active-site tyrosine to break the phosphodiester backbone of DNA.

• This attack forms a covalent bond between the tyrosine hydroxyl group (–OH) and the 5′ phosphate of the DNA.

• This leaves a free 3′ hydroxyl on the DNA strand, which is then used in the strand exchange process.

22) What recombinant sites does phage lambda have? What sites does the prophage have?

🔹 1. Phage Lambda (Before Integration)

• The free lambda phage DNA contains:

  • attP → the phage attachment site

---

🔹 2. Bacterial Chromosome (Before Integration)

• The host E. coli genome contains:

  • attB → the bacterial attachment site

---

🔁 3. After Integration (in the Prophage Form)

When the phage integrates into the bacterial genome (via site-specific recombination between attP and attB), two new hybrid sites are created:

Site	Composition
attL ("left")	Half phage DNA + half bacterial DNA
attR ("right")	Half bacterial DNA + half phage DNA

These are called hybrid attachment sites and flank the prophage within the bacterial genome.

---

🧬 Summary Table:

Stage	Recombination Sites Present
Free phage	attP
Host only	attB
Prophage (integrated lambda)	attL and attR

23) What is the purpose of the intasome?

✅ Purpose of the Intasome:

Function	Description
1. Coordinates Integration	Precisely carries out strand transfer, inserting viral DNA into the host genome.
2. DNA Positioning	Holds the 3′ viral DNA ends in the right orientation for insertion into host DNA.
3. Protects DNA Ends	Prevents degradation and maintains viral DNA in an active conformation.
4. Specificity	Helps recognize or prefer target sites in the host DNA.
5. Enables Efficient Catalysis	Brings all necessary enzymatic domains together in one complex for fast reaction.

---

🔬 Found in:

• Retroviruses (e.g., HIV)

• Other integrating elements, like transposons

• The term is also used in studies of integrase inhibitors (antiviral drugs)

---

🧪 Summary:

The intasome is the functional machine that inserts viral DNA into host DNA — it is essential for stable viral integration and long-term infection.

24) What are two general types of damage to DNA?

1) single base changes

2) structural distortions

25) Name some causes of single-base changes.

1) Replication errors

2) Deamination of cytosine to uracil

26) What are examples of structural distortions?

1) Base analogs

2) Thymine dimer

3) Depurination

4) Alkylation

27) Consider the mutation C/G → A/G. What happens if the cell does not detect the damage and replicates the DNA?

semiconservative mutation

= one cell inherits the original sequence

= other inherits a permanent mutation

28) What are the four steps of nucleotide excision repair?

1) Incision

2) Excision

3) synthesis

4) Ligation

29) Can different repair mechanisms be used to fix one mistake?

Yes

30) What is one difference between glycosylases and lyases?

One difference is glycosylases break glycosidic bonds in nucleic acids while lysases break other types of bonds w/o hydrolysis

31) What is most characteristic about error-prone repair?

It uses low fidelity DNA polymerases that lack proofreading, leading to a higher rate of mutation

32) When a cell is damaged by oxidative stress and 8-oxo-dGTP is produced. Which of the following act on integrated G=O: MutT, MutM, or MutY? Which act on the unintegrated G=O: MutT, MutM, or MutY?

integrated G=O: MutM

integrated G=O, MutT

33) What is one mechanism whereby the cell identifies a newly replicated strand of DNA so it knows which base (causing a mismatch) should be removed?

methylation pattern of DNA

34) What would be the methylation status of GATC sites before replication? After replication?

before: fully methylated

after: hemimethylated

35) Which system tends to remove more nucleotides before the problem is fixed: mismatch repair with the use of GATC or nucleotide excision?

Mismatch repair can remove about 1000 bp, excision can remove up to 10 bp

36) What is the name of the enzyme that usually fills the gap after a piece of DNA strand is removed?

DNA polymerase fills in gap and ligase seals backbone

37) What is the name of the family of genes involved in mismatch repair?

Mut family

38) On what type of region does DNA polymerase slippage usually occur?

polynucleotide track of sequence

39) Which of the mechanisms would be bet at restarting stalled replication forks?

Recombination

40) What can cause double-stranded breakage of DNA?

High energy light rays like UV or x-ray wavelength light

41) In any of these repair systems, is mRNA or a protein used as a template to fix DNA?

No. Only DNA is used to fix DNA.

42) What are three mistakes that the mismatch repair can correct?

• Incorrect base added

• Oxidative damage to DNA

• DNA polymerase slippage

43) As a result of the base flipping mechanism, how many nucleotides will be removed? How many nucleotides are removed during the nucleotide excision repair mechanism?

Base flipping → 1 bp

Nucleotide excision → 10 bp