Volume 20, Issue 5, Pages 783-792 (December 2005) Human DNA Polymerase η Promotes DNA Synthesis from Strand Invasion Intermediates of Homologous Recombination  Michael J. McIlwraith, Alexandra Vaisman, Yilun Liu, Ellen Fanning, Roger Woodgate, Stephen C. West  Molecular Cell  Volume 20, Issue 5, Pages 783-792 (December 2005) DOI: 10.1016/j.molcel.2005.10.001 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 DNA Synthesis on Replication Forks and D Loop Substrates by Human Cell Extracts (A) Schematic representation of the synthetic replication fork. The fork contains a 20 nucleotide primer that can be extended through the 60 base pair duplex to form full-length 80 nucleotide product. The 32P label at the 5′ end of the primer is indicated with an asterisk. (B) DNA synthesis reactions mediated by HeLa whole-cell extract were carried out as described in Experimental Procedures by using 32P-labeled synthetic replication fork DNA. The products were analyzed by denaturing PAGE. The reactions in lanes g–l contained 20 μg extract. (C) Schematic representation of the synthetic D loop structure. The D loop contains a 29 nucleotide primer that can be extended through the 31 base pair duplex to form a 60 nucleotide-long product. The 32P label at the 5′ end of the primer is indicated with an asterisk. (D) DNA synthesis reactions were carried out on 32P-labeled D loops with human HeLa whole-cell extract, and the products were analyzed by denaturing PAGE. T4 and T7 DNA polymerases were used as controls. Lane “a” has no protein. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Fractionation of HeLa Nuclear Extract and Analysis of D Loop Extension Activity (A) Purification scheme is shown. (B) Schematic representation of the synthetic D loop structure used in primer extension assays. The 5′-32P-end label on the primer is indicated by an asterisk. (C–E) Elution profiles from the phosphocellulose, butyl sepaharose, and heparin columns. Aliquots (2 μl) of each fraction were incubated with 32P-labeled D loop substrate, and the products were analyzed by denaturing PAGE. WCE control is indicated. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Identification of polδ and polη in Fractions Exhibiting DNA Extension Activity (A) Peak fractions isolated by column chromatography (Figure 2) were analyzed by western blotting with polɛ, polδ, polα, and PCNA monoclonal antibodies and polι and polη polyclonal antibodies as indicated. (B) Elution of polδ and polη from phosphocellulose, as detected by western blotting. (C) Elution of the primer extension activity from the same phosphocellulose column is shown. Aliquots (2 μl) of each fraction (as indicated) were analyzed by using the 32P-labeled D loop substrate, and the products were analyzed by denaturing PAGE. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Actions of polδ and polη on D Loops and Replication Fork Substrates 32P-labeled D loops (A) or replication forks (B) were incubated with increasing amounts (pmol) of purified human polymerase as indicated. Reaction products were analyzed by denaturing PAGE. The 5′-32P labels on the priming strands are indicated. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 DNA Binding by Purified polη DNA binding assays were carried out as described in Experimental Procedures by using the indicated substrates and amounts of polη. Protein-DNA complexes were analyzed by neutral PAGE. 5′-32P labels are indicated with asterisks. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Extracts from XP-V Cell Lines Are Impaired in Their Ability to Promote Primer Extension from the D Loop Substrate 32P-labeled D loops (A) or replication forks (B) were incubated with 4 μg HeLa or polη-defective XP-V extracts. At the indicated times, the reactions were stopped and DNA products deproteinized and analyzed by denaturing PAGE. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Stimulation of polη-Mediated D Loop Extension by RAD51 (A) 32P-labeled D loops were incubated with polη (15 pmol) and the indicated amounts of human RAD51 protein. DNA synthesis products were analyzed by denaturing PAGE. Lane “a” has no protein. (B) Coimmunoprecipitation of polη and RAD51 from cell extracts prepared from UV-irradiated HeLa S3 cells by using protein A beads coupled to polη polyclonal antibody. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 8 Model Indicating a Dual Role for polη at Stalled Replication Forks (A) A replication fork stalls at a DNA lesion due to the inability of polδ to promote lesion bypass. The 3′ ends of the newly synthesized strands are indicated with arrows. (B) Monoubiquitination of PCNA induces a “polymerase swap,” and polδ is replaced by polη. (C) Polη promotes translesion synthesis to insert a base opposite the lesion and allow replication to proceed. In subsequent reactions, polymerase swap occurs and polδ takes over DNA replication to reestablish normal fork progression. The DNA lesion may now be removed by nucleotide excision repair. (D) As an alternative to lesion bypass, the fork may regress to form a “chicken foot” structure that can undergo nucleolytic cleavage (indicated by arrows). Enzymes involved in this process may include either a Holliday junction resolvase or a flap endonuclease such as MUS81/EME1. Fork regression allows subsequent nucleotide excision repair processes to remove the blocking lesion. (E) The double-strand break formed by fork collapse serves as a substrate for RAD51 binding and the initiation of homologous recombination repair. RAD51 is indicated schematically (blue filament). When the lesion is present on the leading strand, 5′→3′ resection at the DSB will be required to form an extended 3′ single strand for RAD51 binding and subsequent HRR. (F) RAD51 mediates the formation of a D loop that serves as a primer for DNA synthesis by polη. RAD51 may play an active role in polη recruitment. In subsequent steps, the crossover strands need to be cut to reestablish a replication fork. These reactions may involve the MUS81/EME1 endonuclease. Upon reestablishment of the replication fork, polymerase swap occurs and polη is replaced by polδ (C). The displaced strand at the D loop may then serve as a template for lagging-strand synthesis. Molecular Cell 2005 20, 783-792DOI: (10.1016/j.molcel.2005.10.001) Copyright © 2005 Elsevier Inc. Terms and Conditions