Term 2 Practice question: ds DNA breaks during replication

Question 1

Question

Answer 1

Figure 1 shows two situations where a DNA double-strand break could occur during DNA replication. Explain what events might be responsible for creating such a break and how bacterial recombination enzymes could facilitate resumption of DNA replication. You may wish to use diagrams to illustrate how this occurs.

Figure 1. Formation of a double-strand break as a result of DNA replication past a nick or gap in parental duplex DNA (A) or due to a block in fork progression that results in fork reversal and enzyme processing (B).

Question 2

Answer

Answer 2

Answer should initially explain the events that might lead to a single-strand break in parental DNA.

In Fig. 1A (this would most likely arise by oxidative damage, exposure to ionizing radiation, unrepaired nick or gap) or in Fig. 1B (a block to DNA replication – could be a lesion in the DNA or proteins trapped on the duplex).

Better answers could include how fork reversal might occur (either by RecG, RecQ or RuvAB).Should then note that RuvC assembly and symmetrical resolution will generate the break that allows for recombination-dependent replication to be initiated.

Both breaks are essentially the same from this point and need to be processed in exactly the same way. Restoration of this single-ended DNA double-strand break requires the activities of RecBCD (as ahelicase-exonuclease modified by chi-sites to generate 3’-tailed ssDNA), RecA (in homologous pairing and strand exchange) and RuvABC (in branch migration and resolution of Holliday junctions).

Better answers will include a diagram that shows strand invasion, formation of a Holliday junctionand appropriately-orientated resolution to allow replication restart.

Bonus marks for mentioning material not covered in lectures such as the action of primosome assembly via PriA or other pathways to allow reassembly of DNA polymerase III.