Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

Functional class	S. cerevisiae	H. sapiens
End resection	Mre11-Rad50-Xrs2	MRE11-RAD50-NBS1
	Sae2	CtIP
	Exo1	EXO1
	Dna2-Sgs1-Top3-Rmi1	DNA2-BLM-TOP3α-RMI1-RMI2
Adaptors	Rad9	53BP1, MDC1
	–	BRCA1
Checkpoint signaling	Tel1	ATM
	Mec1-Ddc2	ATR-ATRIP
	Rad53	CHK2
	Rad24-RFC	RAD17-RFC
	Ddc1-Mec3-Rad17	RAD9-HUS1-RAD1
	Dpb11	TOPBP1
Single-stranded DNA binding	Rfa1-Rfa2-Rfa3	RPA1-RPA2-RPA3
Single-strand annealing	Rad52	RAD52
	Rad59	–
Mediators	–	BRCA2-PALB2
	Rad52	–
Strand exchange	Rad51	RAD51
	Rad54	RAD54A, RAD54B
	Rdh54	–
Rad51 paralogs	Rad55-Rad57	RAD51B-RAD51C-RAD51D-XRCC2-XRCC3
	Psy3-Csm2-Shu1-Shu2	RAD51D-XRCC2-SWS1
Antirecombinases	Srs2	FBH1, PARI
	Mph1	FANCM
	–	RTEL
Resolvases and nucleases	Mus81-Mms4	MUS81-EME1
	Slx1-Slx4	SLX1-SLX4
	Yen1	GEN1
	Rad1-Rad10	XPF-ERCC1
Dissolution	Sgs1-Top3-Rmi1	BLM-TOP3α-RMI1-RMI2

Open in a separate windowThe vast majority of cell biological studies of mitotic recombination in living cells are performed by tagging of proteins with genetically encoded green fluorescent protein (GFP) or similar molecules (Shaner et al. 2005; Silva et al. 2012). In this context, it is important to keep in mind that an estimated 13% of yeast proteins are functionally compromised by GFP tagging (Huh et al. 2003). By choosing fluorophores with specific photochemical properties, it has been possible to infer biochemical properties, such as diffusion rates, protein–protein interactions, protein turnover, and stoichiometry of protein complexes at the single-cell level. To visualize the location of specific loci within the nucleus, sequence-specific DNA-binding proteins such the Lac and Tet repressors have been used with great success. Specifically, tandem arrays of 100–300 copies of repressor binding sites are inserted within 10–20 kb of the locus of interest in cells expressing the GFP-tagged repressor (Straight et al. 1996; Michaelis et al. 1997). In wild-type budding yeast, such protein-bound arrays are overcome by the replication fork without a cell-cycle delay or checkpoint activation (Dubarry et al. 2011). However, the arrays are unstable in rrm3Δ and other mutants (Dubarry et al. 2011). More pronounced DNA replication blockage by artificial protein-bound DNA tandem arrays has be observed in fission yeast, which is accompanied by increased recombination and formation of DNA anaphase bridges (Sofueva et al. 2011). Likewise, an array of Lac repressor binding sites was reported to induce chromosomal fragility in mouse cells (Jacome and Fernandez-Capetillo 2011). However, these repressor-bound arrays generally appear as a focus with a size smaller than the diffraction limit of light, which is in the range 150–300 nm for wide-field light microscopy.